Liquid Chromatography: Still Liquid Chromatography: Still Striving for High EfficiencyStriving for High Efficiency

Drexel UniversityDrexel University

Literature SeminarLiterature Seminar

byby

Anna CaltabianoAnna Caltabiano

17 May 201017 May 2010

OutlineOutline

IntroductionThe Chromatographic process – theoretical considerationsVariables that affect column efficiencyExisting approaches to improve LC efficiencyConclusionsReferences

IntroductionIntroduction

What is Chromatography?What is Chromatography?

1993, IUPAC formulates: “Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction” [4].

Michael Semenovich Tswett [5]

March 8th, 1903, Tswett emphasizes: “The method is based on the property of dissolved substances to produce physical adsorptive compounds with various mineral and organic solid substances”[1, 2].1906, Tswett names the newly discovered method: “Like light rays in the spectrum, the different components of a pigment mixture are resolved on the calcium carbonate column and then can be qualitatively and quantitatively determined. I call such a preparation a chromatogram, and the corresponding method a chromatographic method” [3].

Why use Chromatography?Why use Chromatography?

Separation of chemical components.Specificity to a particular compound.Purification of chemical components.Identification of chemical components.Quantification of chemical components.Great versatility (a variety of separation modes).

Chromatography todayChromatography todayMore than sixty variants of the technique have been developed.HPLC, GC, SFC, and CE are the most frequently used.HPLC [5]:

almost universal wide range of equipment and columns is commercially available well-understood separation mechanisms sensitive, selective, precise and robust easy to maintain instrumentation flexible in optimizing separations less efficient than some of the separation techniques

GC is preferred to HPLC for analysis of gases, however approximately 75% of all known compounds cannot be separated by GC[5].SFC is higher efficiency than HPLC, but it is limited to non-polar molecules when carbon dioxide is used.Capillary Electrophoresis (CE) is a rival to HPLC [5], nevertheless the detection sensitivity is much lower than in HPLC.

Liquid Chromatography (HPLC) instrumentLiquid Chromatography (HPLC) instrument

Solvent deliverySolvent delivery

PumpPump

AutosamplerAutosampler

ColumnColumn

DetectorDetector

InjectorInjector

The The ChromatographiChromatographi

c Process – c Process – Theoretical Theoretical

ConsiderationsConsiderations

Elution in Column ChromatographyElution in Column ChromatographyMobileMobilephasephase

StationaryStationaryphasephase

tt00 tt11 tt22 tt44tt33

[5][5] [6][6]

Intermolecular InteractionsIntermolecular Interactions

a)a) Dispersion Dispersion (induced dipole-induced dipole)(induced dipole-induced dipole)

δδ--δδ++δδ++

δδ-- δδ++

δδ--

b) Polar b) Polar (dipole-dipole, dipole-induced dipole)(dipole-dipole, dipole-induced dipole)

δδ--δδ++

e) Ionice) Ionic

H

c) Hydrogen bondingc) Hydrogen bonding

NO2NO2

d) Charge transferd) Charge transfer

ChromatogramChromatogram

ColumnColumn

TimeTime

Chromatogram

Chromatogram (Cont)Chromatogram (Cont)A

U

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

a)a)

b)b)

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Minutes

15.45 15.50 15.55 15.60 15.65 15.70 15.75 15.80 15.85 15.90 15.95 16.00 16.05 16.102200-2-2

W = 4σW = 4σ

W0.607 = 2σW0.607 = 2σ

W0.5 = 2.354σW0.5 = 2.354σ

[7]

[8]

[9]

Ideally, the eluted peak would have the same form as the graphical representation of one dimension probability function of the normal distribution of random errors (i.e. Gaussian function) [10].

The chromatogram represents the variation, with time, of the amount of the sample in the mobile phase recorded by the detector.

Retention RelationshipsRetention RelationshipsA

U

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

t0t0

tRtR

[7]

tsts

t0 – void volume; tR –retention time, ts – time spent in the mobile phase; L – column length;u0 – superficial linear velocity of mobile phase; u – the average superficial linear velocity of analyte;VR – retention volume; F – flow rate; R – the fraction of the molecules present in the mobile phase at any time

Plate Number and HETPPlate Number and HETPA

U

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Minutes

15.45 15.50 15.55 15.60 15.65 15.70 15.75 15.80 15.85 15.90 15.95 16.00 16.05 16.10

[7]

[11]

W0.1W0.1

BBBBAAAA

[7]and reduced plate height:

The band broadening process is measured by the column efficiency or plate number N:

The efficiency of chromatographic columns increases with the increase in plate number, while plate height becomes smaller.

The variance per unit length of column can be used as a measure of efficiency:

Peak CapacityPeak Capacity

For complex multi-component compounds the relative performance of separation efficiency is measured by peak capacity instead of plate number.The peak capacity is a hypothetical quantity, which is the total number of peaks that can fit side-by-side from start to finish in a chromatogram.

For isocratic separations:[5]

kmax is retention factor of the last peak; tg is the gradient time; w is the base peak width that is assumed to be constant

For gradient separations: [12]

For small molecule, the peak capacity increases with gradient time and column length in a non-linear fashion. The optimal flow rate for maximum peak capacity is higher than that for maximum theoretical plates.

Variables that Variables that Affect Column Affect Column

EfficiencyEfficiency

Band Broadening and Mobile Phase Band Broadening and Mobile Phase VelocityVelocity

The column efficiency is a measure of band broadening.Peak width and retention time determine plate number:

Various processes that occur inside and outside the column contribute to the peak width:

and

d is contributions from longitudinal diffusion; sm – from stagnant mobile phase mass-transfer, s – from stationary phase mass-transfer, e – from eddy diffusion; m – from mobile phase mass transfer.

[8]

Band Broadening and Mobile Phase Band Broadening and Mobile Phase Velocity (Cont)Velocity (Cont)

Eddy Diffusion (a). Molecules undergo several diversions as they travel along the column. The lengths of the pathways differ significantly [12]. a) Eddy Diffusion

Mobile phase

Particle

b) Mobile Phase Mass-Transfer

Particle

Mobile Phase Mass-Transfer(b). Laminar flow between particles is faster in the stream center than it is near particles [5].

Band Broadening and Mobile Phase Band Broadening and Mobile Phase Velocity (Cont)Velocity (Cont)

Stagnant Mobile Phase and Stationary Phase Mass-Transfer (c). The rate of diffusion of the analyte molecules between the mobile phase outside the pores of the particles (flowing mobile phase) differs from the rate of diffusion within the pores of the particles (stagnant mobile phase) [13].

c) Stagnant Mobile Phase Mass-Transfer

d) Longitudinal diffusion

Longitudinal Diffusion (d). Solutes migrate from a more concentrated part of a medium to a more dilute region [14].Extra-column contributions to band broadening. 1) volumetric - results from the volume of connecting tubing from the injector to the detector, the injection volume and the detector volume; and 2) time-related events - sampling rate and detector time constant [14].

Band Broadening and Mobile Phase Velocity (Cont)Band Broadening and Mobile Phase Velocity (Cont)

The magnitude of band broadening depends on the flow rate (the time of contact between the mobile phase and the stationary phase).This relationship can be described by the van Deemter, the Giddings, the Knox, and several other equations.

Van Deemter plot [15]

Column Length, Column Diameter and Particle SizeColumn Length, Column Diameter and Particle Size

η is the viscosity of the mobile phase ; K is the column permeability; dc is the column internal diameter.

Efficiency can be increased by increasing column length or decreasing particle diameter:

However, these approaches are limited by pressure drop, P, across the column [8].Pressure drop is proportional to column length and inversely proportional to the particle diameter [5, 8]

In theory, efficiency should not depend on column diameter, but practically it has been observed that narrower-diameter columns are usually less efficient that larger-diameter ones [5]. Narrower peaks are more susceptible to extra-column dispersion [21].

[8] [5]

TemperatureTemperatureAt the chemical equilibrium, the relationship between the equilibrium constant and the standard Gibbs free-energy:

Temperature is an important variable in HPLC as it significantly impacts retention factor k:

Since viscosity is decreased at higher temperatures, pressure drop is decreased as well (allows higher flow rates at the same pressure):

Temperature increase promotes better diffusivity and mass transfer of the solutes (increases the molecular diffusion term, B-term, and decreases the mass-transfer term, C-term of the van Deemter equation).

Higher flow rates should be used in order to maintain the same efficiency at the elevated temperatures [14]. Increasing flow rates, in turn, will result in an increase in pressure. The advantage of the elevated temperature is the reduction in the analysis time with negligible loss in column efficiency.

[24]

[25]

kB is Boltzmann constant, d is the diameter of the solute

Existing Existing approaches to approaches to

improve LC improve LC efficiencyefficiency

High Temperature Liquid Chromatography (HTLC)High Temperature Liquid Chromatography (HTLC)

HTLC, operation at temperatures 40 - 200º C, was first proposed by Antia et al. [26]. Theoretically increase in column temperature should provide increase in efficiency. However, practical results were thus far contradictory. In some instances, increase in column temperature led to improvement, had no effect [27, 29], or even decreased column efficiency [28, 29]. Guiochon [30] postulated that the possible cause of theoretical vs. practical discrepancy are radial temperature gradients across the column.Such temperature gradients should be kept to a minimum (not more than a few ºC) since even small gradients will cause large efficiency loss. The temperature of incoming mobile phase should be controlled to prevent “thermal mismatch”.

High Temperature Liquid Chromatography (HTLC) High Temperature Liquid Chromatography (HTLC) (Cont)(Cont)

At high temperatures some compounds may undergo rapid degradation in the column. In some instances decreasing analyte’s residence on the column may and changing the type of the stationary phase may improve the peak shape.The stationary phase has to be stable for an extended period of time. Stationary phase and column hardware might be subject to differential expansion which would result in the formation of voids and channels in the packing material and lead to additional band broadening [33].The mobile phase should be cooled down right after it elutes from the column and before it enters the detector, because the sensitivity of the detector is affected by the temperature of the mobile phase. Possible future of HTLC is miniaturization.

Monolithic Stationary Phase Particles

This approach is based on reduction of the mass-transfer resistance by increasing the external porosity and the flow-through pore size of the packing.A monolithic column consists of a single piece of solid material that consists of interconnected skeletons and interconnected flow paths [35]. The high porosity allows high permeability and thus low pressure drop.Small-sized skeletons of the material offer high efficiency [37]. Heterogeneity of both the silica skeleton and the through-pores determines the efficiency of monolithic columns [39]. The efficiency of polymer-based columns is lower than the efficiency of silica-based monoliths [39].

[35]

Monolithic Stationary Phase Particles (Cont)

Some drawbacks of silica-based monoliths include peak asymmetry (tailing), limited variety and dimensions of monolithic columns [40], limited stability at high pressures, temperatures and pH [41].Tanaka et.al hopes that in the future the operation of monolithic columns on CEC and UHPLC would be possible.

[38]

Fused-Core Stationary Phase Particles

Fused-core particle technology was introduced by Kirkland in 1992 [42]. The approach is based on reduction of the diffusion distance of analytes to minimize stagnant mobile phase mass-transfer.

The shorter diffusion path reduces axial dispersion of solutes and minimizes peak broadening. Reduction in axial dispersion enables higher flow rates without sacrificing column performance.Fused-core particles are characterized by their narrow particle size distribution.

[43] [44]

Fused-Core Stationary Phase Particles (Cont)

Zheng et al.[46]compared the efficiency of totally porous (3 μm), fused-core and monolithic silica based C18 columns for a pharmaceutical product, Celestoderm-V Ointment (an API and critical degradants).

[46]

Small-diameter (sub-2-μm) totally porous stationary phase particles and UHPLC

This technology is based on reduction of the distance that a solute would travel within the stationary phase. This would minimize eddy diffusion (A-term) and the contribution of mass-transfer in the stagnant mobile phase (C-term). When the particle size is reduced, the minimum plate height is decreased and the optimum velocity is increased.

[16]

Small-diameter (sub-2-μm) totally porous stationary phase particles and UHPLC (Cont)

When the particle size is reduced, the backpressure is increased.MacNair et al. [48] predicted theoretical plates, analysis time, and column pressure drop as a function of particle diameter for a small organic analyte with a mobile-phase diffusion coefficient of 6.7 x cm²/s and a capacity factor of 2 1.7um porous particle (Waters UPLC BEH) [44]

[47] [48]

where is the flow resistance factor related to the column packing structure

Φ is the flow resistance factor related to the column packing structure

Small-diameter (sub-2-μm) totally porous stationary phase particles and UHPLC (Cont)

At high flow rates, the friction of the mobile phase against the stationary phase generates heat . The rate of heat generation (power dissipation) is the result of pressure drop and flow rate:

The heat, convectionally transported by the mobile phase along the column, conducts in the axial and radial directions, and evacuates into the air through the column walls and end-fittings [49].

[47]

[49]

Small-diameter (sub-2-μm) totally porous stationary phase particles and UHPLC (Cont)

The axial temperature gradient has only minor consequences since the longitudinal distribution of the mobile phase viscosity along the column affects the local pressure gradient that decreases along the column [49].Since the detector measures the average concentration in the effluent from the column, and the radial concentration profiles of the analytes form an arc in the column (radial band velocity profile) the loss of efficiency is observed.The maximum amplitude of the radial temperature gradient (ΔT) between the column wall and its center:

The frictional heating effect can be reduced by [50, 51]: decreasing the column diameter; not controlling the temperature of the column wall and allowing the column wall to be in

contact with air; cooling the temperature of the entering mobile phase.

At high flow rates using shorter columns should be used for better performance.At optimal flow rates using longer columns will yield very high efficiency.

[47]

q is the heat flux, Rc the column radius, and λ is the radial heat conductivity of the column packing

ConclusionsConclusions

Conclusions

The following approaches have been explored in order to improve HPLC efficiency and speed of analysis: high temperature LC, and monolithic, fused-core, sub-2-μm totally porous column particles technology. The limitations of the HTC technique are uncertainties in column stability and possible on-column degradation of analytes. Monolithic columns offer high efficiency and high speed due to high porosity and small skeleton size, which allow operation at high flow rates on longer columns. However, monolithic columns have limited stability at higher pressures, temperatures and pH. In addition, the limited variety of monolithic columns does not meet the demands for a wide range of industrial applications. Sub-2-μm totally porous particle technology offers high efficiency and shorter analysis times, however it comes at cost of the high pressure. Fused-core technology is relatively young, but it has already demonstrated the great potential in obtaining high efficiency. This technology allows to maintain approximately 80% of the efficiency of totally porous particles at half of the back-pressure, and thus can be used as an alternative to columns packed with sub-2-μm totally porous particles.Overall, the manifested trend of HPLC technology towards smaller particle sizes of stationary phases and narrower column inner diameters clearly indicates the direction of future HPLC development towards miniaturization (HPLC-chip technology).

Thank You!Thank You!

Tswett’s column

Waters Acquity BEH (UPLC)column Agilent HPLC-Chip

ReferencesReferences

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References (Cont)References (Cont)

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References (Cont)References (Cont)

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References (Cont)References (Cont)

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References (Cont)References (Cont)

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Questions?Questions?

Backup SlidesBackup Slides

Band Broadening and Mobile Phase Velocity (Cont)Band Broadening and Mobile Phase Velocity (Cont)

The van Deemter equation (1956) [17] is the simplest, and the most popular equation. It assumes that H consists of three independent contributions that add up. [16]

[16]

[17]

[18]

Knox (1977) introduced equation that describes the relationship between the reduced plate height h and reduced velocity [18].

Giddings (1965) proposed that some portions of the packed-bed dispersion and the mass transfer should be coupled to each other [17].

Band Broadening and Mobile Phase Velocity (Cont)Band Broadening and Mobile Phase Velocity (Cont)

There’s no agreement between scientists which of the equations is the most accurate.Kirkup et al. [19] suggested that the van Deemter equation was the least successful to fit the experimental data, and the Knox equation was just slightly better. Usher et al.[20] opposed Kirkup’s statements with his experimental data that demonstrated the absence of the curvature at high flow rates, and was fit well with the van Deemter equation.

Comparison of equations (Eq. 1 - van Deemter,Eq. 2 - Knox, Eq. 3 - Golay-Gouchon, Eq. 4 - new empirical eq.) [19]

Packing Materials (porous vs nonporous) Packing Materials (porous vs nonporous) and efficiency of packingand efficiency of packing

Nonporous particles provide higher efficiency than porous particles due to faster mass transfer (absence of sample diffusion in the stagnant mobile phase within the pores).The efficiency difference diminishes significantly when the particle size is reduced (to 1.5μm).Porous provide higher sample loading capacity, as well as higher average retention factors [22].Nonporous packings are highly sensitive to extra-column broadening effects [23].Porous particles provide better separation for the early eluting peaks; while nonporous particles provide better separation for the late eluting peaks.

Comparison of van Deemter plots for porous and non-porous particles [22]

Packing Materials (porous vs nonporous) and Packing Materials (porous vs nonporous) and efficiency of packing (Cont)efficiency of packing (Cont)

De Villiers et al. [16] observed increased reduced plate heights (higher than expected C-term) on Acquity 1.7 μm, 2.1 mm internal diameter column.He ascribed this phenomenon to extra-column band broadening, frictional heating effects and poor column packing efficiency associated with complications with producing and packing 1.7 μm particles in a 2.1 mm internal diameter column. Increased bed heterogeneity results in an increase in the A-term and C-term constants which in turn increase reduced plate heights [21]. Comparison of Knox plots for different

particle sizes [16]

Temperature (Cont)Temperature (Cont)

Temperature increase promotes better diffusivity and mass transfer of the solutes (increases the molecular diffusion term, B-term, and decreases the mass-transfer term, C-term of the van Deemter equation).

Temperature increase decreases k, and increases linear velocity. Elevation of column temperature while operating at constant flow rate below 0.4mL/min (below Van Deemter optimum) will result in the loss of efficiency.Higher flow rates should be used in order to maintain the same efficiency at the elevated temperatures [14]. Increasing flow rates, in turn, will result in an increase in pressure. The advantage of the elevated temperature is the reduction in the analysis time with negligible loss in column efficiency.

[25]

kB is Boltzmann constant, d is the diameter of the solute

Temperature (Cont)Temperature (Cont)

Yang [25] proposed a model for temperature effect on column efficiency (under condition of constant linear velocity):

Mass transfer term dominates the separation process at lower temperature.The longitudinal diffusion term, dominates the separation process at higher temperature.Thus, increasing temperature decreases efficiency in the lower temperature range (60 - 100º C), and increases efficiency in the high temperature range (100 - 160º C) [25].

a, b and c are constants

Fused-Core Stationary Phase Particles (Cont)

Cunliffe et al. [47] compared fused-core particles to sub-2-μm totally porous particles (five column chemistries: Waters Acquity BEH C18, 1.7 μm; Agilent Zorbax Extended C18, 1.8 μm; Thermo Hypersil Gold, 1.9 μm; Halo C18 2.7 μm; and Supelco Acentis Express C18, 2.7 μm.) Fused-core particles (Halo and Ascentis Express) can be used as an alternative to columns packed with sub-2-μm totally porous particles.

Van Deemter plot for naphthalene [47]

Fused-Core Stationary Phase Particles (Cont)

Fused-core particles maintain 80% of the efficiency of totally porous particles at half of the back-pressure. This indicates that fused-core particles columns can be coupled to achieve high efficiency [47].Gritti et al. [58] evaluated a new brand of fused-core columns, Kinetex-C18, manufactured by Phenomenex (1.9 μm diameter solid core covered by 0.35 μm porous shell).Kinetex-C18 exhibited HETPs as low as 1.1. This is due to significant improvements in particle size distribution, uniformity of shell thickness, spherical and smooth particle external surface, compared to Halo-C18 columns.These new fused-core particles are characterized by reduction in the eddy diffusion (A-term) and a lower mass-transfer term (C-term) between the mobile and the stationary phase.

Small-diameter (sub-2-μm) totally porous stationary phase particles and UHPLC (Cont)

The amplitude of these gradients depends on the degree of the column's thermal insulation. The longitudinal temperature gradient is the greatest when the column temperature is radially uniform. This happens when there is no radial heat loss through the wall .The radial temperature gradient is the greatest when the column wall temperature is kept constant [49, 50]. Normally, columns are operated under intermediate conditions of thermal insulation where radial and axial temperature gradients co-exist [49]. Temperature gradients have a negative impact on the column efficiency due to heterogeneous distribution of the mobile phase linear velocity, viscosity and density throughout the column, which affect the equilibrium constant of a compound distribution between the mobile and stationary phases (decreases with increasing temperature), and retention factors of the analytes. The difference between the temperatures at the inlet and the outlet of the column can be as much as 20K, and the difference between the temperatures of the center and the column wall can reach up to 6K [51].