ARTICLE pubs.acs.org/ac
Comparison between the Efficiencies of sub-2 μm C18 Particles Packed in Narrow Bore Columns Jesse O. Omamogho and Jeremy D. Glennon* Innovative Chromatography Research Group, Irish Separation Science Cluster (ISSC), Department of Chemistry and the Analytical and Biological Chemistry Research Facility (ABCRF), University College Cork, Ireland ABSTRACT: The chromatographic performance of two types of core-shell particles and two fully porous particles packed in 2.1 ID 50 mm columns was investigated. Comparisons of the performances of the EiS-150-C18 to that of the Kinetex-1.7 μm-C18, Acquity-BEH-1.7 μm-C18, and Zorbax-XDB-1.8 μm-C18 are made and discussed. The physical factors that govern the performance of these columns, such as particle size distribution and column external, total, and particle porosity of the C18 packing materials were among the prime foci of investigation. The differences in the mass transfer behavior measured using naphtho[2,3-a]pyrene between these columns provides an indication of improved performance of the new EiS-150-C18 column. The minimum reduced height equivalent to a theoretical plate (HETP) value for the EiS-150-C18, hmin = 1.95, was achieved and was comparable to that obtained from the C18 phases of the Kinetex (hmin = 2.53), the Acquity (hmin = 2.26), and the Zorbax (hmin = 2.57) columns. This study reveals the importance of the dimension of the shell thickness in controlling the performance of columns packed with shell particles in narrow bore columns.
H
igh performance liquid chromatography (HPLC) column technology is constantly advancing, and the demands for high performance and rapid separation are beginning to be realized with the help of innovative instrument design. The emergence of shell particles in recent times has been a major advance in column technology, prompting the need to readdress the conventional understanding of fundamentals of chromatography. The work of Guiochon and co-workers1-8 have enlightened chromatographers to understand the correlation between the inherent physical properties of the shell particle and the mass transfer phenomena that take place in columns packed with shell particles. A recent study has shown that columns packed with shell particles having the same overall particle diameter, but varied in the ratio of solid core to shell particle, strongly influences the mass transfer kinetic of the columns.9 The performances of columns packed with sub-2 μm coreshell particles are inferior to those packed with the sub-3 μm coreshell particles based on separate studies.6,8 The reduced plate heights provided by the 4.6 mm ID column packed with the Halo-2.7 μm (h = 1.5) and the Kinetex-2.6 μm (h = 1.1) have made these two columns manufactured by Advanced Material Technology and Phenomenex, respectively,8 the best performance columns ever made. However, such performances are negated when these landmark particles are consolidated in narrow bore columns. Fekete et al.10 have shown that the landmark performance of columns packed with the Kinetex-2.6 μm particles is only limited to the standard bore column ID (i.e., 4.6 mm), however, when packed in a narrow bore column (2.1 mm ID), the reduced plate height of 1.9 was the minimum achieved. This suggests that the packing of narrow bore columns does not provide comparable packed bed homogeneity to that of the standard bore columns. Gritti and Guiochon6 studied the r 2011 American Chemical Society
mass transfer kinetics of the Kinetex-1.7 μm-C18 packed in a 2.1 mm ID column, and the minimum reduced plate height was above 2.0. This provides further suggestion that the problematic situation of packing narrow bore columns is compounded when the packing materials are finer such as the sub-2 μm particles. The authors compared the performance of the Kinetex-1.7 μm-C18 to that of fully porous particles (Acquity-BEH-1.7 μm-C18) packed in a 2.1 mm ID column6 at a maximum pressure of 1200 bar. The reduced plate heights were comparable among these columns; however, the Kinetex-1.7 μm-C18 revealed higher A-term compared to the Acquity-BEH-1.7 μm-C18 column.6 The effects of extra-column volume on the performance of narrow bore columns are quite severe, and up to nearly 180% of the efficiency can be lost.11 This gets worse when shell particles with very small porous volumes are packed in narrow bore columns.9 Another factor that deteriorates the performance of sub-2 μm particles consolidated in narrow bore and long columns is the heat friction effect at high flow rate/high back-pressure.12,13 This creates another problem by making the radial dissipation of heat very important, and particles with poor conductivity such as the hybrid particle consisting of organic/inorganic material suffers more from such effect.14,15 This study takes into perspective the performances of selected commercially available sub-2 μm C18 phases packed in 2.1 mm ID columns and compares them to a shell particle column made inhouse. The core-shell C18 column made in-house was also packed in a 2.1 ID 50 mm column and referred to as EiS-150-C18.9 This Received: August 18, 2010 Accepted: January 4, 2011 Published: February 03, 2011 1547
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’ EXPERIMENTAL SECTION Material Characterization. The external surface morphology was measured using a high resolution Inspect F50 (FEI Company Europe) scanning electron microscope (SEM) at 20 kV. An average of five measurements of the particle size distribution of the sub-2 μm particles studied was taken on the basis of electric sensing zone (ESZ) using the Multisizer 3 Coulter Counter enhanced by the digital pulse processor (DPP). Chromatographic data were recorded using the Agilent 1200 RRLC system; data points for the height equivalent to a theoretical plate (HETP) for each column were fitted on the basis of the van Deemter equation. Reagents and Chemicals. All chemicals and reagents were used as supplied from the manufacturers. Octadecyldimethylchlorosilane (97%), imidazole (99%), HPLC grade methanol (99.9%), and naphtho[2,3-a]pyrene (98%) were purchased from SigmaAldrich (Dublin, Ireland). Test mixtures were purchased as separate standards including; uracil (99%), acetophenone (99.5%), benzene (99.9%), toluene (99.8% anhydrous), and naphthalene (þ99% scintillation grade), all from Sigma Aldrich (Dublin, Ireland). Polystyrene standards kits (GPC; MW = 475; 1920; 3250; 10 250; 24 000; 32 500; 67 500; 160 000; 295 000; 705 000; 1 000 000; 2 180 000) were also purchased from Sigma Aldrich (Dublin, Ireland). Deionized water was obtained from a Milli-Q water purification system (Millipore) with resistivity of 18.2 MΩ.cm. Columns. The Kinetex-C18-1.7 μm column (2.1 50 mm) was purchased from Phenomenex, Cheshire, UK. The Acquity-BEHC18-1.7 μm column (2.1 50 mm) was purchased from Waters, (Dublin) Ireland, and the Zorbax-XDB-1.8 μm column (2.1 50 mm) was purchased from Agilent, Waldbronn, Germany. Procedures. Synthesis of C18 Bonded Phases and Column Packing. The C18 bonded phase was prepared in-house on the EiS-150C18 silica particle using monofunctional octadecyldimethylchlorosilane ligand (C20H43Si1Cl1) under controlled reflux condition.16 The EiS-150-C18 was packed in a narrow bore column (2.1 I.D 50 mm) using the low viscosity slurry packing technique.17 Column Porosity Measurement. The resulting packed column porosity was measured by the pycnometry method using pure dichloromethane (DCM) and tetrahydrofuran (THF) to obtain the total column porosity (∈t). The external porosity, ∈e, was measured by inverse size exclusion chromatography (ISEC) using a dilute concentration of 12 polystyrene standards in THF of known molecular weights, injected independently into the column and eluted with pure THF at a flow rate of 0.1 mL/min. The elution volume of the nonretained polystyrene standards was employed to calculate the ∈e from the plot of elution volume vs the cube root of the molecular weight of polystyrene standards. Column Permeability Measurement. The plot of flow rate vs pressure drop was measured for each column to provide a means to estimate the permeability constant of each column. The flow was performed at different flow rates from 0.05 mL/min to a flow rate where the maximum pressure drop is ∼600 bar using a mobile phase of 80% acetonitrile in water. The flow study was performed at a temperature of 295 K, and the pressure at each flow rate was recorded after subtracting the extracolumn backpressure (i.e., replacing the column with a zero dead volume union connector).
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Column Evaluations and Measurement of HETP Data. Chromatography studies were performed to evaluate the separation performance of the EiS-150-C18, the Acquity-BEH-1.7 μm-C18, Kinetex1.7 μm-C18, and the Zorbax-XDB-1.8 μm-C18 on 2.1 I.D 50 mm columns. The chromatographic separation of mixtures of nonpolar small molecules was evaluated on the two shell particle columns (EiS150-C18 and the Kinetex-1.7 μm-C18) and two fully porous particle columns (Acquity-BEH-1.7 μm-C18 and Zorbax-XDB-1.8 μm-C18), under identical mobile phase composition and conditions (50/50 v/v % acetonitrile/water, T = 295 K, and at flow rate of 0.4 mL/min). The following test solutes were used: (1) uracil (90 μg/mL), (2) acetophenone (150 μg/mL), (3) benzene (1500 μg/mL), (4) toluene (6500 μg/mL), and (5) naphthalene (500 μg/mL). An injection volume of 0.3 μL was used, and detection at UV = 254 nm with a sampling rate of 80 Hz and a peak response time of 20 ms were used. A dilute sample of naphtho[2,3-a]pyrene in pure acetonitrile was employed to obtain the HETP data for the measurement of the kinetic performance of the EiS-150-C18 and the commercially packed columns. The mobile phase was 80% acetonitrile in deionized water. Employing the Wilke and Chang equation,18 estimates of the molecular diffusion coefficient of naphtho[2,3-a] pyrene (extended to the mixtures of eluents for the HETP study for each column), is given: pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi xACN ψACN MACN þxH2 O ψH2 O MH2 O Dm ¼ 7:4 10-8 T ð1Þ ηVA 0:6 where VA is the molar volume of naphtho[2,3-a]pyrene at its boiling point (VA = 230.2 cm3/mol), estimated on the basis of the Schroeder and Le Bas method as updated by Sastri et al.,19 MACN = 41 g/mol and MH2O = 18 g/mol are the molecular weights of acetonitrile and water, ψACN = 1 and ψH2O = 2.6 are the solvent association factors for acetonitrile and water, xACN and xH2O are the molar fractions of acetonitrile and water in the mobile phase, respectively, η is the viscosity of the mobile phase (η = 0.54) for a mixture of acetonitrile/ water (80/20 v/v% at 295 K, respectively), and T is the temperature in Kelvin. It is noteworthy that the Dm of naphtho[2,3-a]pyrene is expected to vary widely at a flow rate that generates the maximum pressure drop (600 bar) of the instrument. Such variation is caused by the change in viscosity of the mobile phase due to heat generated inside the column at high inlet pressure. Hence, a true estimate of Dm is achieved, taking into account the heat rise and the decrease in viscosity at high inlet pressure such as 600 bar. For this reason, the Dm given in this report is only a rough estimate. The peak profiles were acquired at frequencies of 10-80 Hz and response rates of 100-20 ms using the Agilent 1200 RRLC UV detection at 298 nm. The eluted peak from the columns was recorded, and the peak variances in the time domain (the second central moment) were estimated using the Foley-Dorsey based model20 of the Exponentially Modified Gaussian (EMG) for all flow rates employed according to the following equation: σtotal 2 ¼
W0:1 2 r =t f Þ2 -11:15ðt r =t f Þþ28 1:764ðt0:1 0:1 0:1 0:1
ð2Þ
W0.12 is the peak width at 10% of the peak maximum, tr0.1 and tf0.1 are the peak width from the center to the rear of the peak and peak width from the center to the front of the peak at 10%, respectively. A similar variance is also estimated when the column was replaced with a zero volume connector to provide 1548
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the extra-column variance. σ ex 2 ¼
W0:1ex 2 2 r f r f 1:764ðt0:1ex =t0:1ex Þ -11:15ðt0:1 =t0:1 Þþ28 ex ex
ð3Þ
Subtracting eq 3 from eq 2 gives the corrected variance of the chromatographic peak eluting from the columns. σ col 2 ¼ σtotal 2 -σex 2 ð4Þ The obtained plate heights for each column were measured by injecting naphtho[2,3-a]pyrene according to the following equation: σ col 2 L ð5Þ H¼ ðtR -tR ex Þ2 where tRexis the retention time of the solute eluting from the zero connector tubing. The extra-column peak variance (σ2ex) given as μL2 was measured according the following equation: σ2ex ¼ Fv 2σ: ex 2
ð6Þ
where Fv, is flow rate (μL/min). The variance of the Agilent 1200 RRLC was 4.8 μL2 using the mobile phase of 80% acetonitrile in water.
’ RESULTS AND DISCUSSIONS Particle Structure and Surface Roughness. Scanning electron microscope (SEM) images of the four packing materials studied are shown in Figure 1A-D. The images reveal the intrinsic surface morphology of these materials, particularly the surface roughness and sphericity. Figure 1A is the SEM images of the 1.7 μm Acquity-BEHC18 particles showing a high degree of sphericity with smooth external surface. Figure 1B is the Kinetex-1.7 μm-C18 particles revealing a slightly rougher surface than the BEH particles but with less sphericity than the former. The SEM image of the 1.8 μm Zorbax particles (Figure 1C) shows a large extent of surface roughness in comparison to the rest of the particles in this study. Furthermore, the Zorbax particles consisted of a rougher surface as shown from the SEM images (Figure 1C). The SEM images of the EiS-150-C18 particles are given in Figure 1D; unlike the Zorbax and Kinetex particles, the SEM image (Figure 1D) reveals a characteristic uniform sphericity and smoother surface. Particle Size Distribution. The particle size distribution measured by the Coulter method revealed quite another interesting property of these sophisticated packing materials as given in Figure 2A-D. The particles of the Zorbax and Acquity columns possesses the largest finite-bandwidth of the particles size distribution, having a d90/10 ratio of 1.83 and 1.57, respectively, translating to a relative standard deviation of 33 and 27% (Figure 2A,B). In contrast, the Kinetex and the EiS-150 provided d90/10 ratios of 1.15 and 1.11, respectively (Figure 2C,D), translating to a relative standard deviation of 6.1 and 5.2%, respectively. This indicates that the EiS-150 is composed of the narrowest particle size distribution. Column Porosity. The two major porosities of the chromatographic columns (characterized by the total and external porosity) were measured; the method employed for this study is described elsewhere in detail.3,15 The total porosity of the Kinetex column (∈t = 0.572) seems to be the largest among the four columns studied; it was slightly larger than the Acquity column packed with fully porous particles (∈t = 0.562). In the first instance, this may seems quite unusual as the Kinetex column is expected to have a smaller total porosity when taking into account the presence of the
Figure 1. Scanning electron microscopy images of the C18 particle studied. (A1) Collection of 30 particles of Acquity-BEH-1.7 μm C18, (A2) single particle showing the external surface, (B1) collection of 30 particles of Kinetex-1.7 μm-C18, (B2) high magnification image revealing external surface, (C1) collection of 30 particles of Zorbax-XDB-1.8 μm-C18, (C2) single particle showing the external surface, (D1) collection of 35 particles of 1.7 μm EiS-150-C18, and (D2) single particle showing the external surface. Note the rugose surface of Zorbax particle and the smoothness of the external surface of the Acquity and EiS particles.
solid core. This unusual scenario can be explained due to the presence of a rough external surface of the Kinetex particles, resulting in a larger external porosity compared to the Acquity column. The Zorbax column has the smallest total porosity (∈t = 0.474) among the three commercial brands of sub-2 μm particles, while the EiS-150 has the smallest total porosity (∈t = 0.448) among all the columns studied. The EiS-150-C18 column has the smallest total porosity and is due to the small porous shell layer (44% by volume to the total particle volume). However, the small ∈t observed for the Zorbax column consisting of totally porous particles is due to the small interparticle porosity ∈p. The external porosity ∈e of each column was measured from the corresponding peak elution volume of polystyrene (Vps), according to following equation: ð7Þ V PS ¼ πrC 2 L∈t ½1þK The extrapolation of the exclusion branch of the ISEC plot to the MW1/3 axis = 0, shown in Figure 3, gave the external volume Vext of the column; ∈e is measured according to the following equation: Vext ð8Þ ∈e ¼ VC where VC is the volume of the empty column used for packing the materials and is given as 0.1732 cm3 (for 2.1 50 mm column). As expected from the SEM images of these particles, the Zorbax1.8 μm-C18 was characterized with the largest external porosity ∈e = 0.421 due to the large extent of surface roughness. In contrast, 1549
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Figure 2. Coulter counter particle size distribution analysis for a collection of 3000 particles. (A) Zorbax-XDB-1.8 μm, (B) Acquity-BEH-1.7 μm, (C) Kinetex-1.7 μm, and (D) EiS-150-1.7 μm. (E) An overlay of the four materials described above. Note the narrow particle size distribution of the EiS particles and the pronounced bimodal distribution of the Zorbax-1.8 μm particles; the size shown on the graph reflects the mode of the particles, i.e., the most frequently observed particle diameter.
the Acquity column has ∈e = 0.381 and the EiS-150-C18 has ∈e = 0.392, indicating the columns packed with these materials are composed of particles of smoother external surface. The Kinetex has a slightly larger external porosity (∈e = 0.405) and is considered to be within the normal range of a spherical packed bed. The particles porosity ∈p for each column was calculated from data obtained from the ∈t and ∈e according to the following equation: ∈p, o ¼
∈t -∈e 1-∈e
ð9Þ
where ∈p,o, is the porosity measured for the columns packed with fully porous particles. Taking into account the volume of solid core present in the particles of the Kinetex and EiS-150-C18 columns, the shell particle porosity is given as ∈t -∈e ð10Þ ∈p, shell ¼ ð1-∈e Þð1-F3 Þ where ∈p,shell is the particle porosity of the shell and F is the ratio of the solid core diameter to the shell particle, and is given as 0.82 and 0.72 for the EiS-150-C18 and the Kinetex-1.7 μm-C18, respectively. 1550
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The shell porosities ∈p,shell of these two columns are 0.208 and 0.449, respectively. The relative porous volume fraction of the shell of the Kinetex-C18 and EiS-150-C18 is 1.41-fold; however, the relative shell porosity is 2.16-fold larger for the Kinetex than the EiS-150-C18. This clearly indicates that the shell pore volume of the Kinetex particles is relatively larger in comparison to the EiS-150-C18. The Zorbax and Acquity columns have ∈p = 0.094 and 0.290, respectively, which suggest that the interparticle porosity of the particles of Zorbax column consists of relatively small pore volume after C18 modification. Table 1 shows the physical characteristic of the four columns studied. Column Permeability. The permeability data is given in Figure 4, showing the plot of column pressure ΔP vs flow rate (mL/min) to obtain the permeability constants of these columns according to the general law of Darcy: Fv ηL ð11Þ ko ¼ ΔPπRc 2 where η, the viscosity of the mobile phase, is given as 0.53 cP, Rc is the radius of the internal column, given as 0.105 cm, and L is the length of the column, given as 5 cm. Because ko is a constant, the flow rate (Fv) and pressure drop (ΔP) selected should give the same value of ko, except at high pressure where eluent viscosity begins to change due to solvent compressibility. At the high inlet pressure, the viscosity of the mobile phase reduces due to heat generation and affects the diffusivity of the solutes. This in turn will provide a local variation of the diffusion coefficient at high inlet pressure; hence, it is desirable to account for this heat generated in the column. For this reason, the flow rate chosen to measure the permeability constant was based on a pressure drop below 200 bar; at such pressure, the change in viscosity due to heat generation is minimized significantly. A flow rate of 0.3 mL/min was used for all columns to measure ko. Table 2 shows the permeability values obtained from each column. From the values of the external porosity, we can estimate the average particle size dp using the Kozeny Carman relationship:21 dp 2 ¼ ko
Figure 3. Plot showing the ISEC measurement of elution volume of 12 polystyrene standards eluted with pure tetrahydrofuran (THF) versus the cubic root of their average molecular weights (MW1/3). Four 2.1 50 mm columns were studied. (A) Acquity-BEH-1.7 μm-C18, (B) Kinetex-1.7 μm-C18, (C) Zorbax-XDB-1.8 μm-C18, and (D) EiS-1501.7 μm-C18. The broken line is an extrapolation at MW = 0 from the excluded ISEC branch and provides a means to determine the external volume and ultimately the external porosity using eq 5.
ð1-∈e Þ2 Kc ∈e 3
ð12Þ
where dp2 and Kc, respectively are the particles diameter and the Kozeny Carman constant given as 180. From the measurement of the permeability, the Acquity column tends to show a 4% larger pressure drop than the Kinetex column. On the contrary, the average particle diameter measured from the ISEC data and eq 12 suggest that the average particle diameter of the Acquity column is approximately 11% larger than the Kinetex particles (assuming Kc ∼ 180). The explanation for the discrepancy in the measurement of the relationship between the permeability and the average particle diameter obtained from eqs 11 and 12, respectively, is due to the dependence of the external porosity in eq 12. The external porosity of the Kinetex and Acquity is 0.405 and 0.381, respectively; thus, it is expected to observe a larger pressure drop from the Acquity column. Another possible explanation may arise from the less dense packed bed of finer particles with rough external surfaces compared to a more dense packing of particles with smooth surface.22 This noncorrelated trend on particle diameter to permeability was severe when comparing the Zorbax column having approximately a 21% smaller pressure drop according to eq 11. However, the average particle diameter is 10% smaller than that of the particles of the Acquity-BEH-1.7 μm-C18 column from the measurement based on eq 12. The external porosity of the 1.8 μm particle Zorbax-XDB-C18 column is 0.421, which results in a smaller pressure drop. The oddity of eq 12 is centered on the 1551
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Table 1. Porosity Data for the EiS-150-C18 Column and the Three Commercially Packed C18 Columnsa Acquity-BEH-C18
Kinetex-1.7-C18
Zorbax-XDB-C18
EiS-150-C18
column batch/serial number
017430035256 37
5574-23/505523-8
B09098/USWEA02187
0000000010
column dimension (mm)
2.1 50
2.1 50
2.1 50
2.1 50
particle diameter (μm)
1.7
1.7
1.8
1.7
total porosityb (∈t)
0.561
0.572
0.474
0.448
external porosityc (∈e)
0.381
0.405
0.421
0.392
particle porosity (∈p,0)
0.290
0.291
0.094
0.092
shell porosity (∈p,shell
0.290
0.449
0.094
0.208
a
Values in parentheses are the shell porosity. b Determined by pycnometry. c Determined by inverse size exclusion chromatography (polystyrene standards).
Figure 4. Plot showing the permeability of four 2.1 50 mm columns as a function of head pressure (bar) vs flow rate (mL/min) from 0.05 up to 1.4 mL/min: Zorbax (empty stars), Kinetex (empty pentagon), Acquity (empty circles), and EiS-150-C18 (cross). The mobile phase is 80% acetonitrile (0.53 cP) in water at temperature of 295 K.
value of the Kc given. For Kc = 180, it gave a close measurement to the Coulter method for a particle characterized of smooth external surface and external porosity close to 0.4. For example, the average particle diameter of the EiS-150-C18 and the Acquity columns that were measured from eq 12 were in good agreement to that measured from the Coulter method. This clearly indicates that the Kc constant for 180 might vary when the external porosity extends above 0.4. A Kc of 250 has been reported for the Halo column that consisted of particles with external porosity of 0.432.1,3 Kinetic Performance of C18 Columns. The extra-column variance (σ2ex) caused by the Agilent 1200 RRLC was measured. The plot of flow rate at 0.05-1.2 mL/min vs the extra-column variance (in volume) of naphtho[2,3-a]pyrene using 80% acetonitrile in water is given by Figure 5. The σ2ex was estimated for each flow rate according to eq 6. Between 0.3 and 1.1 mL/min, the extra-column variance did not exceed 4.8 μL2. The peak variance measured with the columns connected were 13 μL2, 18 μL2, 21 μL2, and 33 μL2 for the EiS-150, Acquity-BEH, Kinetex, and the Zorbax-XDB-C18 columns, respectively (at the maximum flow rates), and corresponded to an extra-column contribution of approximately, 37, 26, 23 and 14% loss of efficiency, respectively. Figure 6 is the van Deemter plot of the reduced HETP vs the reduced linear velocity for the EiS-150-C18 column and three
commercial brands of C18 packing in 2.1 ID 50 mm columns after subtraction of the extra-column broadening due to the instrument. The diffusion coefficient of naphtho[2,3-a]pyrene is 0.89 10-5 cm2/s at T = 295 K, estimated by the Wilke and Chang equation (eq 1). The hmin of 1.92, 2.42, 2.23, and 2.55 were achieved for the EiS-150-C18, Kinetex-1.7-C18, Acquity-1.7-BEHC18, and Zorbax-XDB-1.8-C18, respectively, at the optimum linear velocity (Table 3). It is noteworthy that the mean particle diameter measured by the Coulter (Figure 2) method was taken for the calculation of the reduced HETP. Note that the reduced velocity for all four columns studied were smaller than 12; this means that the van Deemter model gave a practical estimation of the HETP curve shown in Figure 6. It is generally accepted that, at a reduced linear velocity below 20, indistinguishable curves between the van Deemter, Knox, Gidding, and Horvath and Lin equation are observed.23,24 The experimental coefficients of the HETP plots obtained from the EiS-150-C18 and the three commercially C18 packed columns are given in Table 3. The parameters that contributed to band broadening according to the van Deemter equation revealed some interesting results when comparing the mass transfer properties of the EiS-150-C18 columns over the commercial brand of columns. First, the A-term contribution to band broadening obtained from the Kinetex-1.7 μm-C18 (shell column) was the largest (1.51) among the four columns including the EiS-150-C18 (See Table 3). A large A-term from the Kinetex-1.7 μm-C18 (core-shell) column was reported recently by Gritti and Guiochon,6 when comparing the mass transfer properties to that of the Acquity-BEH-1.7 μm-C18 column. Generally, packing of stationary phase materials in narrow bore columns can be problematic resulting in poor bed stability that can have a severe effect on the A-term. However, the large total porosity measured from the Kinetex column may also suggest why the A-term is larger than the rest of the columns emanating from some large radial local differences in the external porosity of the packed bed. The ISEC measurement confirms that the ∈e of the Kinetex-1.7 μm-C18 column has a normal average packed bed and, thus, does not provide a sound justification based on porosity data whether the packing is problematic or not. An on-column detection of solute band to indirectly determine the radial heterogeneity25 of a narrow bore column would aid in the understanding of this puzzle. The experimental A-term for the Zorbax-XDB-C18 column is the smallest (1.41), suggesting that it is the best packed column for this study. This assumption was made taking into account the generality of the intrinsic surface roughness of the Zorbax class of columns,1 having a rugose surface that tends to improve the packing quality. This can be promoted by the large coefficient of shear friction that takes place between the rugose particles of the Zorbax silica during the slurry packing process, requiring a large amount of 1552
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Table 2. Permeability and Effective Particle Diametera permeability (kv) cm2 (Darcy)
EiS-150-C18
Acquity-BEH-C18
2.63 10-11
2.81 10-11
Kinetex C18
Zorbax-XDB-C18
2.92 10-11
3.53 10-11
flow resistance (Φ)
1073
1084
1065
1092
permeability particle diameter (dp) μmb
1.70
1.87
1.67
1.69
a The column permeability was calculated using the following values: F = 0.005 cm3/s, η = 0.0054 (dyn 3 s)/cm2, L = 5 cm, r = 0.105 cm, Δp = 1.48 108 dyn/cm2 (EiS-150-C18), Δp = 1.39 108 dyn/cm2 (Acquity-BEH), Δp = 1.34 108 dyn/cm2 (Kinetex). and Δp = 1.11 108 dyn/ cm2. b Measured with Kozeny-Carman relationship (Kc ∼ 180).
Table 3. HETP Parameter for Naphtho[2,3-a]pyrene (A, B and C) on EiS-150-C18 Column and the Three Commercial Packed C18 Columns with the Given Optimum Reduced Velocity (vopt), the Minimum Reduced Plate Height (hmin), and Retention Coefficient (k0 ) A
B
C
k0
hmin
vopt
ES-150-C18 Kinetex-1.7-C18
1.44 1.51
1.51 3.89
0.003 0.003
5.61 8.95
1.95 2.53
2.78 3.73
Acquity-1.7-BEH-C18
1.45
2.32
0.015
9.34
2.26
2.98
Zorbax-XDB-1.8-C18
1.41
2.18
0.145
10.89
2.57
2.79
columns
Figure 5. Plot showing the experimental extra-column variances of naphtho[2,3-a]pyrene versus flow rates measured on the Agilent 1200 RRLC system. The mobile phase was 80% acetonitrile in water at T = 295 K. The Foley-Dorsey based EMG in volume was used to measure the extracolumn peak variance according to eq 6.
Figure 6. Comparison between the plots of the reduced HETP, h, versus the reduced linear velocity, v, for naphtho[2,3-a]pyrene for 2.1 50 mm packed columns. (b) black curve: EiS-150-C18 column; (2) red curve: Kinetex-C18; (9) green curve: Acquity-BEH-C18; and (() blue curve: Zorbax-XDB-C18. The particle used for measurement of the reduced terms of the plot is given from the Coulter counter measurements. The mobile phase is 80/20 v/v% of acetonitrile/water at T = 295 K.
stress to be applied to the growing packed bed.22 Because of the roughness on the surface of the particles, less slippage is encountered, thus keeping the strains in the packed bed without a rapid change producing a homogeneous packing22 and ultimately contributing to
a reduced A-term. Whereas the particles with smooth surface tends to slip quickly and results in a rapid change in bed strain, causing a more heterogeneous packing consisting of large variation in the local value of external porosity and resulting in a large A-term. This phenomenon described above truly reflected on the data presented for these four 2.1 50 mm columns composed of EiS-150-C18 and the three commercially packed C18. The EiS-150-C18 and the Aquity-BEH-1.7 μm-C18 have closely similar A-term values (1.44 and 1.45, respectively), and their external surfaces are smooth (Figure 1A,D). Second, the B-term contribution to band broadening on the Kinetex-1.7 μm-C18 was the largest (3.89) among the four columns. The largest shell porosity of the Kinetex-1.7 μm-C18 column (0.449) played a major role to provide the largest coefficient of B-term at low linear velocity. At the region of linear velocity where the B-term is significant, the solute will spend more time inside the column and diffuse axially resulting to distortion of the solute band. A significant proportion of the coefficient of the B-term on the Kinetex-1.7 μm-C18 column spawned from surface diffusion, since the solute hold up time in the particle pores resulted in an increase in the specific adsorption of the solutes to the C18 chain. This phenomenon of surface diffusion is extensively documented in an excellent review by Miyabe and Guiochon.26 The EiS-150-C18 has the smallest B-term coefficient (1.51) among the four columns which do not appear to correlate with the particle porosity measured from the Zorbax-XDB-C18 column (∈p = 0.094 vs. 0.208 for Zorbax-XDB-1.8 μm and EiS-150-C18 columns, respectively). The explanation for the larger B-term on the ZorbaxXDB-1.8 μm-C18 column (2.18) in comparison to the EiS-150-C18 column (1.51) is partly due to surface diffusion that contributes largely to the Zorbax-C18 than the EiS-150-C18 column. To illustrate this point, the surface area of the EiS-150 core-shell particle is 80 m2/g, while that of the Zorbax is 180 m2/g (as supplied from the manufacturer). The solute-surface specific adsorption for the Zorbax-XBD-C18 is far larger than the EiS-150-C18 under the same mobile phase conditions. The tendency for the solute to adsorb and diffuse independently at the adsorbed state on the nonpolar stationary phase is larger on the Zorbax-XDB-C18 than on the EiS150-C18 column, leading to a larger axial dispersion at low flow rates. 1553
dx.doi.org/10.1021/ac102139a |Anal. Chem. 2011, 83, 1547–1556
Analytical Chemistry
ARTICLE
Figure 7. Separation of five test solutes on four 2.1 50 mm packed columns (A) EiS-150-C18, (B) Kinetex-1.7 μm-C18, (C) Acquity-BEH-1.7 μm-C18, and (D) Zorbax-XDB-1.8 μm-C18. Eluent: 50/50 v/v% acetonitrile/water. Flow rate: 0.4 mL/min. T = 295 K. Detection: UV = 254 nm. Sample: (1) uracil, (2) acetophenone, (3) benzene, (4) toluene, and (5) naphthalene.
The Acquity-BEH-C18 column is composed of larger surface area, approximately 180 m2/g. The pore size is the largest (130 Å) for all columns studied (i.e., apart from the Acquity-BEH columns, the rest of the columns have particles with nominal pore size of about 90 Å). The experimental B-term is 2.32, and it is larger than that obtained from the Zorbax-XDB-C18 (2.18) column. The surface areas of the particles of these two columns are identical; however, their pore size and porosity vary significantly. Conclusively, in the pore diffusion where no interactions take place between the solute and the wall of the pores, the diffusivity of solute in the stagnant mobile phase was larger for the Acquity-BEH-C18 than the Zorbax-XDB-C18 column at the low flow rate. This causes an additional band spreading that increases more in the Acquity-BEH-C18 than in the Zorbax-C18 column. The contribution to surface diffusion is assumed to be the same for these two columns for the simple fact that the surface area is almost the same. Third, the C-term coefficients for the two shell columns (EiS150-C18 and the Kinetex-1.7-C18) were the smallest. This effect can be ascribed to the presence of the solid core on these columns, leading to a shorter solute diffusional path in and out of the intraparticle pore. A large C-term was observed on the Zorbax column and is ascribed to an enhanced film mass transfer resistance emanating from the rugose surface of the particle.1 In summary, the A-term contributed up to 70% of the total HETP on the EiS-150-C18, whereas, it contributed 54, 60, and 65% for the Zorbax, Kinetex, and Acquity columns, respectively. The B-term contributed 45% of the total HETP for the Kinetex column and contributed only 22, 33, and 27% for the EiS-150C18, Acquity, and Zorbax columns, respectively. Finally, the C-term contributed an almost negligible amount of band broadening on the EiS-150-C18 and the Kinetex-1.7 μm-C18 of
