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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;
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
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.
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.
metoprolol
pindolol
alprenolol
o-chloroaniline
labetalol
HPLC Chromatogram
Instrumentation
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
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).
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
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.
Pumps of HPLC
Reciprocating Pump
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.
Interchangeable loops are available to provide a choice of sample sizes ranging from 5 to 2000 /mL.
Load position
Inject position
Load position
Inject position
Inject position
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.
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Many HPLC instruments incorporate an autosampler with an automatic injector. These can inject continuously variable volumes.
HPLC Detector
The function of the detector in HPLC is to monitor the mobile phase as it emerges from the column.
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
single run.
UV-Vis Detector
Absorbance RT
Absorbance RT
PDA
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)
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
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.
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
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 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).
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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
V0
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.
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62
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.
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.
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:
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.
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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.
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.
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?
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?
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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.
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Peak Tailing
Silanol interactions Degradation of stationary phase
COLUMN POLARITIES
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
1.
Partition
2. 3. 4.
Affinity chromatography
Chiral chromatography Hydrophilic interaction chromatography (HILIC) Electrochromatography: Supercritical fluid chromatography (SFC)
The solute represented by the solid circle () is the more strongly retained.
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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
A primary solvent is used as mobile phase. Addition of secondary solvents is to adjust retention time.
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.
Si
Si-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3
C8
Si
Si-CH2-CH2-CH2-CH3
CH3
C4
Si
Si-CH3
CH3
C1
Si
Si-CH2-CH2CH2NH2
Si Si
Si-CH2-CH2CH2CN Si-CH2-CH2CH2-
Endcapping Capping of exposed silanols with short hydrocarbon chains after the primary bonding step
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
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.
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
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
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)
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
Prednisolone
Prednisone
Prednisolone
Prednisone
Prednisolone
Prednisone
R (CH2)2
Phenyl
s,p
London
R
Halide
F, Cl, Br, I
R O
O
-
London, Dipole-Dipole
Ether
R
s,p London, Dipole-Dipole, H-bonding
Nitro
N O
O
Ester
R O R O
R H O
s,p
Aldehyde
s,p
Ketone
R R
s,p
Amino
NH2
s,p
Hydroxyl
OH
High
Carboxylic Acid
O R OH
s,p
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
H R Si (CH2)3 C R H C C C C
H C H H
R Si (CH2)3 C R N
Toluene
Ethyl Benzene
*Note that the choice may represent a compromise. Here, the optimum column for resolution sacrifices speed.
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
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.
N
(CH2)3CH3
Phenol
3-Butylpyridine
(CH2)5CH3
Anthracene
3-Hexylanthracene
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
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
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
Quick Equilibration
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
Recommended bonded phase (silica based materials only) mark one Normal phase Reversed phase silica C18 NH2 C8 CN Ph CN
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