HPLC

Liquid chromatography (LC) is a physical separation technique conducted in the liquid phase.
A sample is separated into its constituent components by distributing between the mobile phase (a flowing liquid) and a stationary phase (sorbents packed inside a column).

HPLC is a modern form of LC that uses small-particle columns through which the mobile phase is pumped at high pressure.

High Performance?
1. high resolving power; 2. speed of separation;

3. continuous monitoring of the column effluent;

4. accurate quantitative measurement; 5. repetitive and reproducible analysis using the same column; and 6. automation of the analytical procedure and data handling.

High Pressure Liquid Chromatography (HPLC)

An in-line detector monitors the concentration of each separated component band in the effluent and generates a trace called the chromatogram

A representation of the dynamic partitioning process of the analytes between the flowing liquid and a spherical packing particle. The movement of component B is retarded in the column because each B molecule has stronger affinity for the stationary phase than the A molecule.
A schematic of the chromatographic process, where a mixture of analytes A and B are separated into two distinct bands as they migrate down the column filled with packing (stationary phase).

A Brief History

Classical LC was first discovered by Mikhail Tswett in 1903 The invention of gas chromatography (GC) by A. J. P. Martin in 1952
The first generation of high-performance liquid chromatographs was in the 1960s

A Brief Introduction

East West Universitys HPLC

Advantages and Limitations of HPLC

A Schematic Diagram of HPLC

Applications of HPLC
Accurate, precise and robust method for quantitative analysis of pharmaceutical products. Monitoring the stability of drugs in formulations with quantitation of any degradation products.

Measurement of drugs and their metabolites in biological fluids.

Determination of partition coefficients and pKa values of drugs and of drug protein binding. Purification of drugs

HPLC Separation of Benzodiazepines

Peaks: 1 = bromazepam; 2 = nitrazepam; 3 = clonazepam; 4 = oxazepam; 5 = flunitrazepam; 6 = hydroxydiazepam (temazepam); 7 = desmethyldiazepam (nordazepam); 8 = diazepam (valium).

Conditions: sample: 40 ng each; column: 3 cm x 4.6 mm i.d.; stationary phase: ChromSphere C18, 1.5 mm (non-porous); mobile phase: 3.5 ml/min; wateracetonitrile (85 : 15); temperature: 350C; UV detector 254 nm.

Typical modern chromatogram of a mixture of b-blockers

propanolol

metoprolol

pindolol

Column: Zorbax Eclipse-XDB C18; Solvent: acetonitrile/water mixture at pH = 3

alprenolol

o-chloroaniline

labetalol

HPLC Chromatogram

HPLC Instrumentation and Techniques

Solvent Delivery Mobile phase reservoirs Construction pump material Recriprocating Pumps Syringe Sample Introduction Manual Injection Automated Injection Packings Column Packings and harware Measure column performance Column care and use UV-Vis Fluorescence Detectors Refractive Index Conductivity Volatametry/Amperometry

Instrumentation

Solvent Delivery System in HPLC

He degasser

Mobile Phase Resorvoir The reservoir that holds the mobile phase is often no more than a glass bottle.
Bottle capped on outside with a UV- absorbing plastic

The reagent bottle that holds HPLC solvent is used as a reservoir. Solvent is delivered from the reservoir to the pump by means of Teflon tubing--called the "inlet line" to the pump

Purpose of a sinker frit

Push filter (10 mm) prevent any dust or particulate matter to enter the pump.

This is because particulate matter can interfere with pumping action, damage valves and seals. In addition, it may also damage the column (by collecting at the top of the column).

Filtration of Mobile Phase of HPLC

Degassing of the liquid mobile phase

DEGASSING The practice of removing air from the mobile phase; degassing can be achieved by bubbling He gas into the M.P

Why is required?
He degassing removes dissolve oxygen from the M.P.
The presence of oxygen in mobile phase causes bubble formation resulting in air in the flow system and pump pressure will change causing spike in the chromatogram (due to air bubble formation in the detector cell)
Instead of He degassing vaccum degassing method can also be used

Requirements for a solvent reservoir

The reservoir and its attachment to the pump should be made of materials that will not contaminate the mobile phase: Teflon, glass, or stainless steel.
The vessel should have some sort of cap to prevent particulate matter from contaminating the mobile phase. If you are using a solvent bottle as a reservoir, the top of the bottle can be wrapped in aluminum foil to keep dust out or the bottle cap can be drilled to allow inserting the inlet line through the cap.

Don't close the bottle too tightly or removal of mobile phase by the pump will create a vacuum.
This prevents mobile phase from flowing the pump, creating a "vapor lock" within the pump.

Filtration of Sample of HPLC

Inner Diameter (ID) of Tubing of HPLC

The actual cross-sectional distance across the open interior of a tube through which mobile phase flows. This is an important parameter in at least three components: connecting tubing, column tubing, injector tubing.

The volume-length relationship is shown in the table below.

Pumps of HPLC

Pumping of Mobile Phase in HPLC System

Reciprocating Pump

Elution of Mobile Phase in HPLC System

Elution of Mobile Phase in HPLC System

Isocratic elution

An HPLC system generating a separation under conditions in which composition of the mobile phase does not change with time.
composition of the solvent is changed continuously or in a series of steps.

Gradient elution

A gradient denotes that there is a parameter in the separation that is changed with time in order to affect the elution. In HPLC a gradient refers to a change in the mobile-phase composition with time. Gradient profiles can be linear, step, nonlinear, convex and concave.

Gradient vs. Isocratic Elution in HPLC System

Low Pressure Gradient Elution in HPLC System

High Pressure Gradient Elution in HPLC System

Injector of HPLC System

Interchangeable loops are available to provide a choice of sample sizes ranging from 5 to 2000 /mL.

Load/Inject Position of Injector

Load position

Inject position

Load/Inject Position of Injector

Load position

Inject position

Load/Inject Position of Injector

Load position

Inject position

Sample Injecting Syringes of HPLC

Injector and Loop of HPLC

Filling the Loop by Autosampler

Filling the loop by three different autosampler designs: A, pull-loop; B, push-loop; C, integral-loop injection. 5 = to column, 6 = to waste, 7 = lowpressure seal, 8 = high-pressure seal,

1 = sample vial, 2 = syringe with stepper motor, 3 = loop, 9 = position of the movable loop when the valve is switched 4 = from pump and the sample transferred to the column.
35

Injection of Sample by Auto Sampler

Sample Rack Control Rack

Injection of Sample Mechanism by Auto Sampler

Many HPLC instruments incorporate an autosampler with an automatic injector. These can inject continuously variable volumes.

Injection of Sample Mechanism by Auto Sampler

HPLC Detector
The function of the detector in HPLC is to monitor the mobile phase as it emerges from the column.

Cells of HPLC Detector

The detector should be small and compatible with liquid flow. No highly sensitive, universal detector system is available for high-performance liquid chromatography. The detector used will depend on the nature of the sample.

Ultraviolet Detectors
The UV absorption detector is the most widely used in HPLC. It is based on the principle of absorption of UV/visible light as the effluent from the column is passed through a small flow cell held in the radiation beam. It is characterised by high sensitivity (detection limit of about 1 x 10-9 g/mL - for highly absorbing compounds) and it is relatively insensitive to changes of temperature and flow rate. The detector is generally suitable for gradient elution work since many of the solvents used in HPLC do not absorb to any significant extent at the wavelengths used for monitoring the column effluent. The presence of air bubbles in the mobile phase can greatly impair the detector signal, causing spikes on the chromatogram. Both single and double beam instruments are commercially available. Although the original detectors were single- or dual-wavelength instruments (254 and/or 280 nm), some manufacturers now supply variable-wavelength detectors covering the range 210800 nm so that more selective detection is possible.

HPLC Detectors
UV-Vis PDA

PDA Detectors

Photo Diode Array Detector (PDA)

Photodiode array detectors scan a range of wavelengths every few milliseconds and continually generate spectral information. Wavelength, RT and absorbance can all be plotted.

PDA detector provide three dimensional

information that allows an accurate assessment of peak identity, purity and quantitation in a

single run.

Three Dimensional Chromatogram by PDA

UV-Vis Detector

Absorbance RT

Absorbance RT

PDA

Three Dimensional Chromatogram by PDA

Three Dimensional Chromatogram by PDA

Peak purity analysis by 3D Chromatogram of PDA

It is difficult to determine component purity chromatogram. from a

A PDA detector can analyze peak purity by comparing spectra within a peak. A pure peak has matching spectra throughout the peak (at all wavelengths)

Refractive Index Detectors

These detectors are based on the change of refractive index of the eluant from the column with respect to pure mobile phase.

Although they are widely used, the refractive index detectors suffer from several disadvantages lack of high sensitivity, lack of suitability for gradient elution, and the need for strict temperature control ( +/- 0.001oC) to operate at their highest sensitivity

Refractive Index Detectors

In this detector, both the column mobile phase and a reference flow of solvent are passed through small cells on the back surface of a prism. When the two liquids are identical, there is no difference between the two beams reaching the photocell, but when the mobile phase containing solute passes through the cell, there is a change in the amount of light transmitted to the photocell, and a signal is produced.

Fluorescence Detectors
These Detectors enable fluorescent compounds present in the mobile phase to be detected by passing the column effluent through a cell irradiated with ultraviolet light and measuring any resultant fluorescent radiation.

Although only a small proportion of inorganic and organic compounds are naturally fluorescent, many biologically active compounds (e.g. drugs) and environmental contaminants (e.g. polycyclic aromatic hydrocarbons) are fluorescent.
These Detectors have very high sensitivity. Because both the excitation wavelength and the detected wavelength can be varied, the detector can be made selective.

An widespread used detector.

Columns for HPLC

Most HPLC columns are made of 316-grade stainless steel, which is chromiumnickel molybdenum steel, resistant to the usual HPLC pressure and also relatively inert to chemical corrosion. Columns, of i.d. 25mm are generally used for analytical purposes. Wider columns of i.d. between 10 mm and 25.4 mm may be used for preparative work.

Some companies even market preparative columns of i.d. 30 cm and more.

Columns 5, 10, 15 or 25 cm long are common

They are generally made from stainless steel tubing, fittings, and frits.

Column length

Effect on chromatography

Short (30-150mm) - short run times, low backpressure Long (250-300mm) - higher resolution, long run times

Column ID

Narrow bore ( 2.1 mm) - higher detector sensitivity, Sharp peak Analytical 4.6 mm Preparative (10-22 mm) - high sample loading

Chromatographic terminology and Definition

Retention Time
The time between the sample injection point and the analyte reaching a detector is called the retention time (tR).

Rt, T0, V0

Void Time (to).

The retention time of an unretained component (often marked by the first baseline disturbance caused by the elution of the sample solvent) is termed void time (to).

Void Volume (V0).

The volume of solvent eluted during void time is known as the column void volume (V0).

Void Volume
Even if the analyte does not interact with the stationary phase, it will not appear in the detector immediately after injection.
An HPLC column is filled with small particles of porous material which have a significant volume of the liquid phase between the particles and inside their porous space, so the non-interacting analyte still has to travel through this volume before it enters the detector. The volume of the liquid phase in the column is called void volume (V0). Several other names are also used in the chromatographic literature: dead volume, hold-up volume, If a particular HPLC system provides constant and stable mobile-phase flow (F), one can convert retention volume (VR) and void volume (V0) into the retention time (tR) and a void time (t0).

56

Chromatographic terminology and Definition

Void Volume (V0)

The concept of column void volume (V0) is important for several reasons. Void volume is the volume of the empty column minus the volume occupied by the solid packing materials. It is the liquid holdup volume of the column that each analyte must elute from.

V0

Note that the void volume (V0) is equal to the void time (t0) multiplied by the flow rate (F). Vo = t0 F Or

Void volume V0 = 0.65 r2 L where r is the inner radius of the column and L is length of the column.

the

Chromatographic terminology and Definition

Note that V0 is proportional to the square of the inner radius of the column. It is important to have a rough idea of the void volume of the column since it often dictates the 1. operating flow-rate range, 2. sample loading capacity, and 3. mass sensitivity (the minimum detectable amount of the assay). For example, a typical analytical column (150 mm 4.6 mm i.d.) has a V0 of about 1.5 mL and is operated at ~l.0 mL/min. In contrast, by reducing the inner diameter to 2.0 mm, a typical LC/MS column (150 mm 2.0 mm i.d.) has a V0 of about 0.3 mL and is operated at ~0.2 mL/min. Column void volumes also control the volumes of the eluting peaks. Smaller column void volumes lead to smaller peak volumes and, therefore, higher analyte concentration. As a result, if the same mass of analyte is injected, smalldiameter columns lead to higher sensitivity.

V0

Chromatographic terminology and Definition

Wb and h

The peak of HPLC chromatogram has both width (Wb) and height (h). The subscript b denotes that the width was measured at the base line. Sometimes the width halfway up the peak (W1/2) or at 5% of peak height (W0.05) is used to meet the method or compendial requirements.

V0
Void time can be interpreted as part of the total analyte retention time that the analyte actually spends in the mobile phase moving through the column, and for the rest of the retention time the analyte sits on the stationary phase surface. The void volume or void time is a very important parameter, and its correct determination could be critical for the interpretation of the experimental results.

60

BASIC CHROMATOGRAPHIC DESCRIPTORS

Four major descriptors are commonly used to report characteristics of the chromatographic column, system, and particular separation: 1. Retention factor (k) 2. Efficiency (N) 3. Selectivity () 4. Resolution (R)

Retention Factor (k)

The analyte retention consists of two parts: (1) the time the component resides in the mobile phase actually moving through the column and (2) the time the analyte is retained on the stationary phase. The difference between the total retention time (tR) and the hold-up time is called the reduced retention time (tR), and corresponding difference between the analyte retention volume and the void volume is called the reduced retention volume, VR. The ratio of the reduced retention volume to the void volume is a widely used dimensionless parameter called retention factor, k.

62

A Quantitative Description of Column Efficiency

Two related terms are widely used as quantitative measures of chromatographic column efficiency: (1) plate height (H) (2) plate count or number of theoretical plates (N).

The efficiency of chromatographic columns increases as the plate count (N) becomes greater and as the plate height (H) becomes smaller. Enormous differences in efficiencies are encountered in columns as a result of differences in column type and in mobile and stationary phases.

Efficiencies in terms of plate numbers can vary from a few hundred to several hundred thousand.

Plate Theory
Plate theory is used for the evaluation of the column efficiency. Plate theory assumes that the analyte is in the instant equilibrium with the stationary phase and the column is considered to be divided into a number of hypothetical plates. Each plate has a finite height (height of effective theoretical plate, HETP), and an analyte spends a finite time in this plate. This time is considered to be sufficient to achieve quilibrium. The smaller the plate height or the greater the number of plates, the more efficient the analyte exchange is between two phases, and the better the separation.

That is why column efficiency is measured in number of theoretical plates.

Experimental Determination of the Number of Plates in a Column

Xs = K . X m Where Xm is the concentration of solute in the mobile phase, Xs is the concentration of solute in the stationary phase, and K is the distribution coefficient of the solute between the two phases. It should be noted that K is defined with reference to the stationary phase (i.e., K =Xs/Xm), thus the larger the distribution coefficient, the more the solute is distributed in the stationary phase.

Consider three consecutive plates in a column, the p -1, the p, and the p =1 plates and let there be a total of n plates in the column. The three plates are depicted in Figure.

Experimental Determination of the Number of Plates in a Column Column Efficiency (N)

In most chromatograms, peaks tend to be Gaussian in shape and broaden with time, where Wb becomes larger with longer tR. This is caused by band-broadening effects inside the column, and is fundamental to all chromatographic processes.
The term, plate number (N), is a quantitative measure of the efficiency of the column, and is related to the ratio of the retention time (tR) and the standard deviation of the peak width (Wb).

theoretical plates

Experimental Determination of the Number of Plates in a Column

Number of theoretical plates,

Following chromatogram shows a peak width (wb) of 10 units and a tR of 135 units. The column efficiency (N) can therefore be calculated as follows:

Experimental Determination of the Number of Plates in a Column

The plate theory shows that the peak width (the dispersion or peak spreading) is inversely proportional to the square root of the efficiency and, thus, the higher the efficiency, the narrower the peak.
68

Selectivity
The ability of the chromatographic system to discriminate different analytes is called selectivity (a). Selectivity is determined as the ratio of the retention factors of two analytes, or the ratio of the reduced retention times

The increase of the selectivity in the development of the separation of a complex mixture is the primary goal of any chromatographer, because if the selectivity for the pair of analytes is equal to 1, then it does not matter how narrow your peaks or how fast your separationyou will not be able to separate these components until you increase the selectivity.

Selectivity vs. Eficency

I. Peaks are narrow and far from each other, simple decrease of the column length or flow rate can significantly shorten the runtime without the loss of separation quality. II: Acceptable separation, method may not be rugged. III: Acceptable separation, quantitation reproducibility could be low. IV: Bad separation.

70

?
A chromatographic analysis for the chlorinated pesticide Dieldrin gives a peak with a retention time of 8.68 min and a baseline width of 0.29 min. How many theoretical plates are involved in this separation? Given that the column used in this analysis is 2.0 meters long, what is the height of a theoretical plate?

the number of theoretical plates is

Solving equation for H gives the average height of a theoretical plate as

71

Resolution
Resolution, R, is defined as the ratio of the distance between two peaks to the average width of these peaks at baseline. This descriptor encompasses both the efficiency and selectivity.

The resolution (R) of a column tells how far apart two bands are relative to their widths.
The resolution provides a quantitative measure of the ability of the column to separate two analytes.

72

Factors Influencing HPLC Separation

Parameters affecting efficiency:
Flow rate Column length Particle diameter Particle size distribution

Parameters affecting retention factor:

Eluent type Eluent composition Stationary phase type Analyte nature

Parameters affecting selectivity:

Stationary phase type Analyte nature Eluent additives Temperature Eluent composition (ionizable analytes)

Peak Tailing
Silanol interactions Degradation of stationary phase

Unswept void volume, or void formation at head of column,

Co-eluting material POOR MATCH BETWEEN ANALYTE, MOBILE PHASE, AND

COLUMN POLARITIES

Tailing Factor (Tf)

Front width

Peak width

5% height

Tf = (peak width) / 2 x (fronts half width) All widths measured at 5% peak height. Values greater than 1.5 generally indicate that unwanted interactions are occurring.

Peak Fronting

Overloaded column. Channels in the solid phase.

Retention Mechanisms Applicable to HPLC

Four retention mechanisms are applicable to HPLC:

1.

Partition

2. 3. 4.

Adsorption Ion exchange and Exclusion

Which Methods of HPLC

Modes of HPLC Normal-Phase Chromatography (NPC) Reversed-Phase Chromatography (RPC)

Ion-Exchange Chromatography (IEC)

Size-Exclusion Chromatography (SEC)

Affinity chromatography
Chiral chromatography Hydrophilic interaction chromatography (HILIC) Electrochromatography: Supercritical fluid chromatography (SFC)

Schematics showing the basis of separation

(a) adsorption chromatography, (b) partition chromatography, (c) ion-exchange chromatography, (d) Size exclusion chromatography.

The solute represented by the solid circle () is the more strongly retained.

80

Silica gel (Adsorption Modes of HPLC)

Progress of a chromatographic separation

Graphical Representation of the Separation Process

(a) A mixture of two components, and is applied to the chromatographic bed.
(b) The component resides for preference in the stationary phase and the component more in the mobile phase .1b). Here k = 5/2 = 2:5 and k = 2/5 = 0:4. (c) A new equilibrium follows the addition of fresh eluent: sample molecules in the mobile phase are partly adsorbed by the naked stationary phase surface, in accordance with their distribution coefficients, whereas those molecules that have previously been adsorbed appear again in the mobile phase. (d) After repeating this process many times, the two components are finally separated. The component prefers the mobile phase and migrates more quickly than the component, which tends to stick in the stationary phase.

Interaction of Normal Phase

Stationary Phase for Normal Phase of HPLC

Si

Si-OH

Unmodified Silica

Si

Si-CH2-CH2-CH2-NH2

Amino

Si

Si-CH2-CH2-CH2-CN

Cyano

OH
Si Si-CH2-CH2-CH2-OCHCH2 OH Diol

Mobile Phase Phase for Normal Phase of HPLC

Primary solvents (non-polar)

Hydrocarbons (Pentane, Hexane, Heptane, Octane) Carbon tetrachloride Aromatic Hydrocarbons (Benzene, Toluene, Xylene) Methylene chloride Chloroform Ethyl acetate, Acetone 2-propaol, ethanol, methanol, Acetonitrile THF, Methyl-t-butyl ether (MTBE), Diethyl ether, Dioxane, Pyridine

Secondary solvents (polar)

A primary solvent is used as mobile phase. Addition of secondary solvents is to adjust retention time.

Partition Modes of HPLC

In liquid-liquid partition chromatography, a solute distributes itself between two immiscible liquid phases in a manner analogous to the partition that occurs in liquidliquid extraction. One liquid phase (the stationary phase) forms a thin film over the surface of an inert support, while the other liquid phase (the mobile phase) passes over the first. Thus, the retention mechanism is the partitioning of solutes between mobile liquid phase and stationary liquid phase.

Stationary Phase for Reversed Phase of HPLC

Stationary Phase for Reversed Phase of HPLC

Stationary Phase for Reversed Phase of HPLC

Column for Reverse Phase

Si Si-CH2-CH2CH2-----CH3 (C18) ODS

Si

Si-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3

C8

Si

Si-CH2-CH2-CH2-CH3
CH3

C4

Si

Si-CH3
CH3

C1

Stationary Phase for Reversed Phase of HPLC

Si

Si-CH2-CH2CH2NH2

Amino Cyano Phenyl

Si Si

Si-CH2-CH2CH2CN Si-CH2-CH2CH2-

Stationary Phase for Reversed Phase of HPLC

Endcapping Capping of exposed silanols with short hydrocarbon chains after the primary bonding step

Mobile Phase for RP

Water + Organic Solvents - When buffer is used, the concentration and pH are important factors Methanol, Acetonitrile or THF are common organic solvents for RP HPLC

Mobile Phase for RP

Increase of Solvent Polarity

Normal Phase vs. Reversed Phase

Interaction of analytes with stationary phase MP vs. RP

Particle Shape

Most modern HPLC packings materials have spherical particles, but some are irregular in shape.
Irregular particles have larger surface areas, and are relatively inexpensive. Spherical particles offer lower backpressure, and higher performance, stability, and reproducibility than irregular particles.

Particle Shape

Smaller particle sizes give higher efficiency and higher resolution than larger particle sizes.
Larger particle sizes offer faster flow rates and lower back-pressure. Typical particle sizes range from 3 mm to 20 mm, and new 1.5 mm particle sizes are available to maximize resolution on short columns. A 5 mm particle size represents the best compromise between efficiency and back-pressure.

Particle Shape

For silica-based reversed-phase packings, carbon load indicates the amount of functional bonded phase attached to the base material.
Phases with lower carbon loads are more weakly hydrophobic, which may significantly reduce retention times over phases with higher carbon loads. However, a higher carbon load will give higher capacity and often greater resolution, especially for compounds of similar hydrophobicity.

Surface Area

Effect on chromatography
High surface area generally provides greater retention, capacity and resolution for separating complex, multicomponent samples. Low surface area packings generally equilibrate quickly, especially important in gradient analyses.

Pore Size

Effect on chromatography
Larger pores allow larger solute molecules to be retained longer through maximum exposure to the surface area of the particles. Choose a pore size of 150 or less for sample MW 2000. Choose a pore size of 300 or greater for sample MW > 2000.

A Note The larger the pore diameter, the smaller the surface area. The larger the surface area the greater the retention. The smaller the pore diameter the greater the steric hindrance effect.

pH stability

The main parameter affecting pH stability of packing material is Bonding Density Low pH (<2.5) causes hydrolysis of the siloxane bonds destroying bonded layer The higher the bonding density the lower hydrolysis effect. High pH (>8.5) causes silica dissolution High bonding density shield silica surface which makes it stable up to pH 13.

Endcapping

Effect on chromatography
Endcapping reduces peak-tailing of polar solutes that interact excessively with the otherwise exposed, mostly acidic silanols. Non-endcapped packing provide a different selectivity than do endcapped packing, especially for such polar samples.

HPLC Method Development

Introduction
Common Mistakes in Method Development: Inadequate Formulation of Method Goals Little Knowledge of Chemistry of Analyte Mixture Use of the First Reversed Phase C18 Column Available Trial and Error with Different Columns and Mobile Phases These Mistakes Result In: Laborious, Time-consuming Development Projects Methods that Fail to Meet the Needs of the Analyst

HPLC Method Development - A Proposed Procedure

At Your Desk Define your knowledge of the sample Define your goals for the separation method Choose the columns to be considered In the Laboratory Choose the initial mobile phase chemistry Choose the detector type and starting parameters Evaluate the potential columns for the sample Optimize the separation conditions (isocratic or gradient) for the chosen column Validate the method for release to routine laboratories

Choosing the Appropriate HPLC Column Should Be Based Both Upon Knowledge of the Sample and Goals for the Separation

Benefits of this Approach Include: Small initial time investment Big time savings in the HPLC laboratory More informed approach to column selection More efficient than trial and error approach

Knowledge of the Sample Influences the Choice of Column Bonded Phase Characteristics
Knowledge of the Sample
Structure of sample components? Number of compounds present? Sample matrix? pKa values of sample components? Concentration range? Molecular weight range? Solubility? Other pertinent data?

Column Chemistry

(bonded phase, bonding type, endcapping, carbon load)

Goals for the Separation Influence the Choice of Column Particle Physical Characteristics
Goals for the Separation Max. resolution of all components? Partial resolution? Fast analysis? Economy (low solvent usage)? Column stability and lifetime? Preparative method? High sensitivity? Other goals?

Column Physics

(particle bed dimensions, particle shape, particle size, surface area, pore size)

Column Selection Chart

Suitable for MW >2000 High Resolution High Sensitivity Default Column (Good for most Applications) High Efficiency Low Backpressure Stability at pH Extremes High Capacity High Stability Fast Analysis Low Mobile Phase Consumption High Sample Loadability Fast Eqilibration

Particle Size small (3m) medium (5m) large (10m) Column Length short (30mm) medium (150mm) long (300mm) Column ID narrow (2.1mm) medium (4.6mm) wide (22.5mm) Surface Area low (200m2/g) high (300m2/g) Pore Size small (60) medium (100) large (300) Carbon Load low (3%) medium (10%) high (20%) Bonding Type monomeric polymeric Particle Shape spherical irregular

Method Goals

Choosing the Bonded Phase

Draw the molecular structures for all known components of the mixture. Identify the two compounds whose structures are the most similar. e.g.:
O HO OH O HO O OH HO

Prednisolone

Prednisone

Choosing the Bonded Phase

For these two molecules, circle the structural features that differ. It is these differences that should be exploited to optimize the separation. e.g.:
O HO OH O HO O OH HO

Prednisolone

Prednisone

Choosing the Bonded Phase

Use the results of the structural comparison to select a bonded phase showing optimal selectivity for these two molecules. In this case consider using a silica column (no bonded phase) for its ability to retain polar solutes through hydrogen bonding.
HO O HO OH O O OH HO

Prednisolone

Prednisone

Functional Group Polarity Comparisons

Polarity Low Functional Group Methylene Structure Bonding Types s Intermolecular Forces Displayed London

R (CH2)2

Phenyl

s,p

London

R
Halide

F, Cl, Br, I
R O
O
-

London, Dipole-Dipole

Ether

London, Dipole-Dipole, H-bonding

R
s,p London, Dipole-Dipole, H-bonding

Nitro

N O

O
Ester

R O R O
R H O

s,p

London, Dipole-Dipole, H-bonding

Aldehyde

s,p

London, Dipole-Dipole, H-bonding

Ketone

R R

s,p

London, Dipole-Dipole, H-bonding

Amino

NH2

s,p

London, Dipole-Dipole, H-bonding, Acid-base chemistry

Hydroxyl

OH

London, Dipole-Dipole, H-bonding

High

Carboxylic Acid

O R OH

s,p

London, Dipole-Dipole, H-bonding, Acid-base chemistry

Examples of bonded phases used for HPLC packing media:

Choosing the Bonded Phase

C18 or Octadecylsilane (ODS) Very nonpolar - Retention is based on London (dispersion) interactions with hydrophobic compounds. Example : Alltima C18

R Si (CH2)17 CH3 R

Choosing the Bonded Phase

Phenyl Nonpolar - Retention is a mixed mechanism of hydrophobic and p - p interactions. Example : Platinum Phenyl

H R Si (CH2)3 C R H C C C C

H C H H

Choosing the Bonded Phase

Cyanopropyl Intermediate polarity - Retention is a mixed mechanism of hydrophobic, dipole-dipole, and p - p interactions. Example : Alltima CN

R Si (CH2)3 C R N

Choosing the Bonded Phase

Each bonded phase has unique selectivity for certain sample types. As a practical example, to separate toluene and ethyl benzene: Note a difference of one -CH2- unit Choose a C18 bonded phase for retention by hydrophobicity Maximize hydrophobic selectivity with a high silica surface area, high carbon load material like Alltima C18

Toluene

Ethyl Benzene

Choosing the Particle Physical Characteristics

Use the Column Selection Chart Use default column as starting point Match up method goals with individual particle physical characteristics Change only those particle parameters that affect the method goals Recognize the optimum column as a possible compromise Example: Sample Type: hydrophobic compounds Method Goal: highest resolution

Choosing the Particle Physical Characteristics Example:

Sample Type: hydrophobic compounds Method Goal: highest resolution
Column Selection Chart Default Column Column Bed Dimensions Particle Size Surface Area Pore Size Carbon Load Bonding Type Base Material Particle Shape 150 x 4.6mm 5m 200m2/g 100 10% Monomeric Silica Spherical Optimum Column 250 x 4.6mm 3* or 5m >200m2/g 100 16 - 20% Mono- or Polymeric Silica Spherical

* mobile phase backpressure may be excessive

Optimum Column: Alltima C18, 5m, 250 x 4.6mm

*Note that the choice may represent a compromise. Here, the optimum column for resolution sacrifices speed.

Choosing the Particle Physical Characteristics

Column Dimensions Length and internal diameter of packing bed Particle Shape Spherical or irregular Particle Size The average particle diameter, typically 3-20m

Surface Area Sum of particle outer surface and interior pore surface, in m2/gram

Choosing the Particle Physical Characteristics Pore Size Average size of pores or cavities in particles, ranging from 60-10,000 Bonding Type Monomeric - single-point attachment of bonded phase molecule Polymeric - multi-point attachment of bonded phase molecule Carbon Load Amount of bonded phase attached to base material, expressed as %C Endcapping Capping of exposed silanols with short hydrocarbon chains after the primary bonding step

Column Dimensions
Effect on chromatography
Column Dimension Short (30-50mm) - short run times, low backpressure Long (250-300mm) - higher resolution, long run times Narrow ( 2.1mm) - higher detector sensitivity Wide (10-22mm) - high sample loading

Particle Shape
Effect on chromatography Spherical particles offer reduced back pressures and longer column life when using viscous mobile phases like 50:50 MeOH:H2O.

Particle Size
Effect on chromatography Smaller particles offer higher efficiency, but also cause higher backpressure. Choose 3m particles for resolving complex, multi-component samples. Otherwise, choose 5 or 10m packings.

Surface Area
Effect on chromatography High surface area generally provides greater retention, capacity and resolution for separating complex, multi-component samples. Low surface area packings generally equilibrate quickly, especially important in gradient analyses. High surface area silicas are used in Alltima, Adsorbospherel HS, and Adsorbosphere UHS packings. Low surface area silicas are used in Alltechs Platinum, Econosphere, and Brava packings.

Pore Size
Effect on chromatography Larger pores allow larger solute molecules to be retained longer through maximum exposure to the surface area of the particles. Choose a pore size of 150 or less for sample MW 2000. Choose a pore size of 300 or greater for sample MW > 2000.

Bonding Type
Effect on chromatography Monomeric bonding offers increased mass transfer rates, higher column efficiency, and faster column equilibration.
CH3 OH + X Si (CH2)17 CH3 CH3
monomeric bonding

R Si (CH2)17 CH3 R

OH + OH X

CH3
polymeric

O
bonding

CH3 Si (CH2)17 CH3

Si (CH2)17 CH3 X

Polymeric bonding offers increased column stability, particularly when highly aqueous mobile phases are used. Polymeric bonding also enables the column to accept higher sample loading.

Carbon Load
Effect on chromatography Higher carbon loads generally offer greater resolution and longer run times. Low carbon loads shorten run times and many show a different selectivity.

Endcapping
Effect on chromatography Endcapping reduces peak-tailing of polar solutes that interact excessively with the otherwise exposed, mostly acidic silanols. Non-endcapped packings provide a different selectivity than do endcapped packings, especially for such polar samples. Platinum EPS packings are non-endcapped to offer enhanced polar selectivity.

Conclusion
In this approach to HPLC column selection, the bonded phase chemistry of the column is chosen on the basis of an analysis of the sample component structures. The physics of the column is chosen according to an analysis of the goals for the separation method. This approach succeeds in predicting unique, optimum bonded phase chemistries and particle bed physical characteristics that are likely to meet the goals for the separation method.

Column Selection Example #1

What goals do I have for the method? Maximum resolution of all components? Best Peak Shape for difficult samples? Fast analysis? Economy (low solvent consumption)? Column stability-long lifetime? Purify one or more unknown components for characterization? High sample loadability? High sensitivity? Other (High Sample Throughput--Quick Equilibration) What do I know about the sample? Number of compounds present Sample matrix pKa values of compounds? UV spectral information about compounds? Concentration range of compounds Molecular weight range of compounds 4 --94 - 323

Column Selection Example #1

Structures of Compounds
OH

N
(CH2)3CH3

Phenol

3-Butylpyridine

(CH2)5CH3

Anthracene

3-Hexylanthracene

Column Selection Example #1

Which two sample components have the most similar structures? Draw them, then circle the structural differences between them. Note: The structural difference between these two compounds is the hydrophobic hexyl side chain. This suggests a non-polar C18 or C8 column would interact with this area of difference to help provide separation of these two compounds.

Anthracene

3-Hexylanthracene

(CH2)5CH3

Recommended bonded phase (silica based materials only) mark one Normal phase Reversed phase silica C18 NH2 C8 CN Ph CN

Column Selection Example #1

Column physical characteristics use Column Selection Chart and Method Goals Column bed dimensions (mm) Particle Size (m) Surface area (m2/g) Pore Size () Carbon Load (%) Bonding type Particle shape Default Column Ideal Column 150 x 4.6 100 x 2.1 5 5 200 <200 100 100 10 10 Monomeric Monomeric spherical spherical

Column Selection Example #1

Available packing alternatives meeting the above criteria:
Packing Base Particle Particle Carbon Pore Surface Material Shape Size Load Size Area (m) (%) () (m2/g) silica Sph. 3, 5, 10 12 80 220 Bonding EndType capd Mono. Yes

Allsphere ODS-2 Brava BDS C18 Econosphere C18 Platinum C18

silica
silica silica

Sph.
Sph. Sph.

3, 5
3, 5, 10 3, 5, 10

8.5
10 6

145
80 100

185
200 200

Mono.
Mono. Mono.

Yes
Yes Yes

Column Selection Example #1

Available packing alternatives meeting the above criteria:
Packing Base Particle Particle Carbon Pore Surface Material Shape Size Load Size Area (m) (%) () (m2/g) silica Sph. 3, 5, 10 12 80 220 Bonding EndType capd Mono. Yes

Allsphere ODS-2 Brava BDS C18 Econosphere C18 Platinum C18

silica
silica silica

Sph.
Sph. Sph.

3, 5
3, 5, 10 3, 5, 10

8.5
10 6

145
80 100

185
200 200

Mono.
Mono. Mono.

Yes
Yes Yes

Best Peak Shape

Increased Sensitivity, Low Solvent Consumption, Fast Analysis

Quick Equilibration

Column of choice: Brava BDS C18, 100x2.1, 5m (Spherical , 185m2/g, monomeric)

Good balance of efficiency & backpressure Reduced backpressure

Column Selection Example #2

What goals do I have for the method? Maximum resolution of all components? Partial resolution, resolving only select components? Fast analysis? Economy (low solvent consumption)? Column stability-long lifetime? Purify one or more unknown components for characterization? High sample loadability? High sensitivity? Other What do I know about the sample? Number of compounds present Sample matrix pKa values of compounds? UV spectral information about compounds? Concentration range of compounds Molecular weight range of compounds

6+ --UV -254 -349 - 645

Column Selection Example #2

Structures of Compounds
N N O N H2N O H H NH N O HO O S O H H NH N H2N O HO O O O O HO O S S N N N S

N O H H NH N

O N

O H H S NH N O HO O

O NH2

N O O N O NH O H NH H S N O HO HO S O N N N N O H NH S O HO O H N S O O

Column Selection Example #2

Which two sample components have the most similar structures? Draw them, then circle the structural differences between them.
Notes: both structures very polar, with amine and pi bond functions--a RP CN column may give good separation by mixed-mode retention of hydrophobic, CN---H---NR2 hydrogen bonding and p-p interactions with double bonds.
O H2N O H H NH N O HO O S H NH S O HO O H N S O O

Recommended bonded phase (silica based materials only) mark one Normal phase Reversed phase silica C18 NH2 C8 CN Ph CN

Column Selection Example #2

Column physical characteristics use Column Selection Chart and Method Goals Column bed dimensions (mm) Particle Size (m) Surface area (m2/g) Pore Size () Carbon Load (%) Bonding type Particle shape Default Column Ideal Column 150 x 4.6 250 x 2.1 5 5 200 200 + 100 Not critical 10 -Monomeric Polymeric spherical spherical

Column Selection Example #2

Available packing alternatives meeting the above criteria:
Packing Base Particle Particle Carbon Material Shape Size Load (m) (%) silica silica silica silica Irreg. Sph. Sph. Sph. 5, 10 3, 5 3, 5, 10 3, 5, 10 ----Pore Size () 60 100 80 100 Surface Bonding EndArea Type capd 2/g) (m 450 350 220 200 Poly. Poly. Mono. Mono. Yes Yes No No

Adsorbosil CN Alltima CN Allsphere CN Platinum CN

High resolution, High sensitivity

High res.

Column of choice: Alltima CN, 250 x 2.1 , 5m ( Spherical , 350 m2/g , polymeric)
Good balance of efficiency & backpressure Reduced backpressure Robust