The impact of extra-column band broadening on the chromatographic efficiency of 5 cm long narrow-bore very efficient columns

Small columns packed with core-shell and sub-2 μm totally porous particles and monolith columns are very popular to conduct fast and efficient chromatographic separations. In order to carry out fast separations, short (2-5 cm) and narrow-bore (2-2.1 mm) columns are used to decrease the analyte retention volume. Beside the column efficiency, another significant issue is the extra-column band-spreading. The extra-column dispersion of a given LC system can dramatically decrease the performance of a small very efficient column. The aim of this study was to compare the extra-column peak variance contribution of several commercially available LC systems. The efficiency loss of three different type 5 cm long narrow bore, very efficient columns (monolith, sub-2 μm fully porous and sub-2 μm core-shell packing) as a function of extra-column peak variance, and as a function of flow rate and also kinetic plots (analysis time versus apparent column efficiency) are presented.     

Kinetic investigation of narrow-bore columns packed with prototype sub-2 μm superficially porous particles with various shell thickness

The recent successful breakthrough of sub-3 μm shell particles in HPLC has triggered considerable research efforts toward the design of new brands of core-shell particles. We investigated the mass transfer mechanism of a few analytes in narrow-bore columns packed with prototype 1.7 μm shell particles, made of 1.0, 1.2, and 1.4 μm solid nonporous cores surrounded by porous shells 350, 250, and 150 nm thick, respectively. Three probe solutes, uracil, naphthalene, and insulin, were chosen to assess the kinetic performance of these columns. Inverse size exclusion chromatography, peak parking experiments, and the numerical integration of the experimental peak profiles were carried out in order to measure the external, internal, and total column porosities, the true bulk diffusion coefficients of these analytes, the height equivalent to a theoretical plate, the longitudinal diffusion term, and the trans-particle mass transfer resistance term. The residual eddy diffusion term was measured by difference. The results show the existence of important trans-column velocity biases (7%) possibly due to the presence of particle multiplets in the slurry mixture used during the packing process. Our results illustrates some of the difficulties encountered by scientists preparing and packing shell particles into narrow-bore columns.     

Core–shell column Tanaka characterization and additional tests using active pharmaceutical ingredients

In the last decade, core–shell particles have gained more and more attention in fast liquid chromatography separations due to their comparable performance with fully porous sub‐2 μm particles and their significantly lower back pressure. Core–shell particles are made of a solid core surrounded by a shell of classic fully porous material. To embrace the developed core–shell column market and use these columns in pharmaceutical analytical applications, 17 core–shell C₁₈ columns purchased from various vendors with various dimensions (50 mm × 2.1 mm to 100 mm × 3 mm) and particle sizes (1.6–2.7 μm) were characterized using Tanaka test protocols. Furthermore, four selected active pharmaceutical ingredients were chosen as test probes to investigate the batch to batch reproducibility for core–shell columns of particle size 2.6–2.7 μm, with dimension of 100 × 3 mm and columns of particle size 1.6 μm, with dimension 100 × 2.1 mm under isocratic elution. Columns of particle size 2.6–2.7 μm were also tested under gradient elution conditions. To confirm the claimed comparable efficiency of 2.6 μm core–shell particles as sub‐2 μm fully porous particles, column performances of the selected core–shell columns were compared with BEH C₁₈, 1.7 μm, a fully porous column material as well.     

Characterization and kinetic performance of 2.1 × 100 mm production columns packed with new 1.6 μm superficially porous particles

The overall kinetic performance of three production columns (2.1 mm × 100 mm format) packed with 1.6 μm superficially porous CORTECS‐C₁₈+ particles was assessed on a low‐dispersive I‐class ACQUITY instrument. The values of their minimum intrinsic reduced plate heights (h_min = 1.42, 1.57, and 1.75) were measured at room temperature (295 K) for a small molecule (naphthalene) with an acetonitrile/water eluent mixture (75:25, v/v). These narrow‐bore columns provide an average intrinsic efficiency of 395 000 plates per meter. The gradient separation of 14 small molecules shows that these columns have a peak capacity about 25% larger than similar ones packed with fully porous BEH‐C₁₈ particles (1.7 μm) or shorter (50 mm) columns packed with smaller core–shell particles (1.3 μm) operated under very high pressure (>1000 bar) for steep gradient elution (analysis time 80 s). In contrast, because their permeabilities are lower than those of columns packed with larger core–shell particles, their peak capacities are 25% smaller than those of narrow‐bore columns packed with standard 2.7 μm core–shell particles.     

Comparison of core-shell versus fully porous particle packings for chiral liquid chromatography productivity

A loading and productivity study was done using three racemates on vancomycin and teicoplanin-bonded chiral stationary phases of different particle formats. Two columns were packed with 2.7 μm superficially porous particles and two columns were packed with identically bonded 5 μm fully porous particles. The last two columns were packed with specially synthesized 4.5 μm vancomycin and teicoplanin superficially porous particles. The loading of different chiral compounds showed that the columns filled with 2.7-μm chiral stationary phases were inappropriate for preparative separations due to their very low permeability which precluded high flow rates. However, columns containing 4.5 μm superficially porous (core-shell) particles were as effective for small-scale preparative chiral separations as columns filled with classical 5 μm fully porous particles. Comparing the 4.5 μm superficially porous particles and 5 μm fully porous particles teicoplanin columns, the observed respective productivities of 270 and 265 mg/g chiral phase/h for 5-methyl-5-phenyl hydantoin enantiomers were obtained. Particular attention was given to the peculiar case of the mianserin enantiomeric separation on vancomycin columns that gave observed productivities of 200 and 205 mg/g chiral phase/h on the 4.5 μm superficially porous particles and 5 μm fully porous particles, respectively.     

Mass transfer mechanism in liquid chromatography columns packed with shell particles: Would there be an optimum shell structure?

The mass transfer mechanisms in columns packed with old (55 μm Zipax and 5 μm Poroshell) and recently commercialized shell particles (2.7 μm Halo-C(18) and Kinetex-C(18)) were investigated from a physico-chemical point of view. Combining a model of diffusion in heterogeneous packed beds (effective medium theory) with values of the heights equivalent to a theoretical plate (HETPs derived from the first and second central moments of the elution profiles) and of the peak variances provided by the peak parking method, we demonstrate that columns packed with current shell particles perform better than those packed with fully porous particles in resolving low molecular weight compounds because the eddy diffusion term of the van Deemter equation of the former is markedly smaller. The calculation of eddy diffusion in column beds suggests that the smaller A terms are due to smaller trans-column velocity bias in columns packed with shell particles. We also show that the mass transfer of large molecules (e.g., proteins) is faster when the internal volume accessible to the analyte increases. Therefore, it is suggested that shell particles made of concentric layers with average pore sizes increasing with increasing diameter would provide columns with higher efficiency.     

Feasibility of ultra high performance supercritical neat carbon dioxide chromatography at conventional pressures

The implementation of columns packed with sub-2 μm particles in supercritical fluid chromatography (SFC) is described using neat carbon dioxide as the mobile phase. A conventional supercritical fluid chromatograph was slightly modified to reduce extra column band broadening. Performances of a column packed with 1.8 μm C18-bonded silica particles in SFC using neat carbon dioxide as the mobile phase were compared with results obtained in ultra high performance liquid chromatography (UHPLC) using a dedicated chromatograph. As expected and usual in SFC, higher linear velocities than in UHPLC must be applied in order to reach optimal efficiency owing to higher diffusion coefficient of solutes in the mobile phase; similar numbers of theoretical plates were obtained with both techniques. Very fast separations of hydrocarbons are presented using two different alkyl-bonded silica columns.     

Some practical comparisons of the efficiency and overloading behaviour of sub-2 μm porous and sub-3 μm shell particles in reversed-phase liquid chromatography

At their optimum flow, sub-3 μm superficially porous or "shell" particles demonstrate similar efficiency to sub-2 μm totally porous particles. The performance of 0.21 cm i.d shell columns is however inferior to those of 0.46 cm i.d., presumably due to packing difficulties. At high flow, shell columns can give flatter Knox curves due to lower operating pressure (half or less of that of the totally porous particles) producing less frictional heating, which combined with the increased thermal conductivity of their non-porous core, gives more efficient heat dissipation. However, the effects of frictional heating for sub-2 μm columns are considerably exaggerated when using pure ACN as mobile phase, as it has a thermal conductivity 3 times less than that of pure water, leading to poorer heat dissipation. Overloading is already problematic for ionised solutes, a group which contains many pharmaceuticals and compounds of clinical relevance, on conventional columns (5 μm porous particles). However, it becomes a more serious issue for both new column types, partially as a result of their very high efficiency, which concentrates the sample as a very narrow band. The sample capacity of one type of shell particle was estimated to be 60% of that of the small totally porous particles, in line with the fraction of the particle volume that is porous. Due to overloading, it is barely possible to achieve perfect peak symmetry for ionised acids or bases with either of these new column types, even by injecting the lowest amounts of sample detectable by UV. While ammonium formate and potassium phosphate buffers gave similar results in overloading studies, use of formic acid as sole mobile phase additive is not recommended for these solutes, as its ionic strength is too low, leading to a catastrophic deterioration in efficiency when sample concentrations of even a few mg/L are injected.     

A simple isocratic LC method for quantification of trace-level inorganic degradation impurities (ferricyanide,ferrocyanide, nitrite,and nitrate) in sodium nitroprusside injection and robustness by quality using design approach

This study developed and validated a trace-level quantification inorganic impurities method using reversed-phase HPLC and performed the robustness check using quality-by-design approach by varying the multiple factors simultaneously. This method is economical and simple and exhibits its stability-indicating nature [for the determination of ferrocyanide ([Fe(CN)₆]^4–), ferricyanide ([Fe(CN)₆]³⁻), nitrate (NO₃^–), and nitrite (NO₂^–)] in sodium nitroprusside (SNP) drug substance and liquid dosage form. Chromatographic separation was achieved using a USP L43 column (ACE PFP, 150 × 4.6 mm, 3 μm) with a simple isocratic elution. The buffer consists of potassium dihydrogen phosphate (50 mM), tetrabutylammonium hydrogen sulfate (9 mM), and tetrabutylammonium hydroxide (25 mM). The buffer pH was adjusted to 7.2 with tetrabutylammonium hydroxide. The mobile phase was mixed with the buffer and acetonitrile (68:32 v/v). The flow rate was 0.8 mL/min, column temperature was maintained at 30°C, and injection volume was 5.0 μL. The SNP impurities were monitored at 225 nm using a UV detector. Further, the method was validated per the International Council for Harmonisation (ICH) guidelines, and forced degradation studies were carried out under different stress conditions. The detector responses were plotted against concentrations, and correlation was linear (r > 0.999) over the range of 0.8–7.5 μg/mL for ferricyanide; 1.0–37.5 μg/mL for SNP; and 0.2–7.5 μg/mL for ferrocyanide, nitrite, and nitrate. The method repeatability was established for all the impurities with relative standard deviation (%), and the results were found to be less than 2.0.     

Kinetic investigation of the relationship between the efficiency of columns and their diameter

Many brands of packing materials made of fine particles are now available in both conventional (4.6 mm i.d.) and narrow-bore (2.1 mm i.d.) columns. It is a general observation that the efficiency of the former tends to be markedly higher than that of the latter. This report provides a detailed illustration of the characteristics of this enigma. The corrected reduced plate heights of three brands of columns packed with shell particles in 4.6 and 2.1 mm I.D. columns were measured. The brands were the 1.7 and 2.6 μm Kinetex-C(18) (Phenomenex, Torrance, CA, USA), the 2.7 μm Poroshell120-C(18) (Agilent Technologies, New Castle, DE, USA), and the 2.7 μm Halo-C(18) (Advanced Material Technologies, Wilmington, DE, USA). The extra-column contributions were minimized by optimizing the configuration of the instrument (injection volume <1.0 μL, 115 μm needle seat capillary, 80 μm connecting tubes, no heat exchanger, 0.8 μL detection cell). The correct peak variances were derived from the numerical integration of the first and second order moments of the experimental band profiles. These experimental results confirm that the kinetic performance of narrow-bore columns is inferior to that of conventional columns for all three brands of shell particles. We demonstrate that this difference is accounted for by a contribution to the column HETP of the long-range eddy diffusion term that is larger in the 2.1 than in the 4.6 mm I.D. columns. While the associated relative velocity biases are of comparable magnitude in both types of columns, the characteristic radial diffusion lengths are of the order of 100 and 40 μm in the wall regions of narrow-bore and conventional columns, respectively.     

Properties and performance of novel high-resolution/high-permeability ion-exchange media for protein chromatography

There is continued interest in the development of stationary phases for protein chromatography that can provide high resolution at elevated flow rates of the mobile phase. When using porous particles, resolution and dynamic binding capacity decline rapidly as the flow rate is increased. Monolithic columns have been developed to overcome these limitations. However, there are difficulties in manufacturing homogeneous larger scale monoliths. In this paper we investigate the morphology and performance characteristics of columns based on new ion exchangers obtained by mechanically disrupting continuous beds of acrylamido-based polymeric media. Near colloidal suspensions of loose particles obtained with this procedure can be flow-packed in ordinary chromatography columns resulting in beds of unexpectedly high hydraulic permeability. Columns up to 2.2 cm in diameter were studied with both Q and S functionalized media. The hydraulic permeability and interparticle porosity of these columns were rather high. The permeabilities of the S and Q media were 1.5 x 10(-13) and 2.4 x 10(-13) m2, respectively, while the corresponding porosities were 60 and 70%. These porosity values are similar to those of monoliths, suggesting that these particles assemble under flow to give high-porosity bridged structures. The structure of these packed beds was further characterized by embedding small packed columns in resins and obtaining sections for microscopic observation. The sections reveal the presence of small aggregates of non-porous 1-3 microm particles, surrounded by flow channels several micrometers in size. The height equivalent to a theoretical plate under isocratic and gradient elution conditions and the dynamic binding capacity were determined for several proteins and were found to be virtually independent of flow.     

Comparison between core–shell and totally porous particle stationary phases for fast and green LC determination of five hepatitis‐C antiviral drugs

The performances of core–shell 2.7 μm and fully porous sub‐2 μm particles packed in narrow diameter columns were compared under the same chromatographic conditions. The stationary phases were compared for fast separation and determination of five new antiviral drugs; daclatasvir, sofosbuvir, velpatasvir, simeprevir, and ledipasvir. The gradient elution was done using ethanol as green organic modifier, which is more environmentally friendly. Although both columns provided very good resolution of the five drugs, core–shell particles had proven to be of better efficiency. Under gradient elution conditions, core–shell particles exhibited faster elution, better peak shape, and enhanced resolution adding to lower system backpressure. The column backpressure on sub‐2 μm particles was more than twice that on core–shell particles. This gives a chance to use conventional high‐performance liquid chromatography conditions without needing special instrumentation as that required for ultra‐high performance liquid chromatography. The method was validated for determination of the five drugs by gradient elution using mobile phase composed of organic modifier ethanol and aqueous part containing 0.75 g sodium octane sufonate and 3.0 g sodium dihydrogen phosphate per liter at pH of 6.15. Detection was done using UV‐detector set at 210 nm. The linearity, accuracy, and precision were found very good within the concentration range of 2–200 μg/mL.     

Polymethacrylate monolithic and hybrid particle-monolithic columns for reversed-phase and hydrophilic interaction capillary liquid chromatography

We prepared hybrid particle-monolithic polymethacrylate columns for micro-HPLC by in situ polymerization in fused silica capillaries pre-packed with 3–5 μm C₁₈ and aminopropyl silica bonded particles, using polymerization mixtures based on laurylmethacrylate–ethylene dimethacrylate (co)polymers for the reversed-phase (RP) mode and [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl) zwitterionic (co)polymers for the hydrophilic interaction (HILIC) mode. The hybrid particle-monolithic columns showed reduced porosity and hold-up volumes, approximately 2–2.5 times lower in comparison to the pure monolithic columns prepared in the whole volume of empty capillaries. The elution volumes of sample compounds are also generally lower in comparison to packed or pure monolithic columns. The efficiency and permeability of the hybrid columns are intermediate in between the properties of the reference pure monolithic and particle-packed columns. The chemistries of the embedded solid particles and of the interparticle monolithic moiety in the hybrid capillary columns contribute to the retention to various degrees, affecting the selectivity of separation. Some hybrid columns provided improved separations of proteins in comparison to the reference particle-packed columns in the reversed-phase mode. Zwitterionic hybrid particle-monolithic columns show dual mode retention HILIC/RP behaviour depending on the composition of the mobile phase and allow separations of polar compounds such as phenolic acids in the HILIC mode at lower concentrations of acetonitrile and, often in shorter analysis time in comparison to particle-packed and full-volume monolithic columns.     

Separation of cannabinoids on three different mixed‐mode columns containing carbon/nanodiamond/amine‐polymer superficially porous particles

Three mixed‐mode high‐performance liquid chromatography columns packed with superficially porous carbon/nanodiamond/amine‐polymer particles were used to separate mixtures of cannabinoids. Columns evaluated included: (i) reversed phase (C₁₈), weak anion exchange, 4.6 × 33 mm, 3.6 μm, and 4.6 × 100 mm, 3.6 μm, (ii) reversed phase, strong anion exchange (quaternary amine), 4.6×33 mm, 3.6 μm, and (iii) hydrophilic interaction liquid chromatography, 4.6 × 150 mm, 3.6 μm. Different selectivities were achieved under various mobile phase and stationary phase conditions. Efficiencies and peak capacities were as high as 54 000 N/m and 56, respectively. The reversed phase mixed‐mode column (C₁₈) retained tetrahydrocannabinolic acid strongly under acidic conditions and weakly under basic conditions. Tetrahydrocannabinolic acid was retained strongly on the reversed phase, strong anion exchange mixed‐mode column under basic polar organic mobile phase conditions. The hydrophilic interaction liquid chromatography column retained polar cannabinoids better than the (more) neutral ones under basic conditions. A longer reversed phase (C₁₈) mixed‐mode column (4.6 × 100 mm) showed better resolution for analytes (and a contaminant) than a shorter column. Fast separations were achieved in less than 5 min and sometimes 2 min. A real world sample (bubble hash extract) was also analyzed by gradient elution.     

Characterization and identification of multiple constituents in Yinhuang granules by high-performance liquid chromatography with diode-array and time-of-flight mass spectrometry detection

A fast high-performance liquid chromatography (HPLC) method with diode-array detection (DAD) and time-of-flight mass spectrometry (TOF/MS) has been developed for the analysis of multi-constituent in Yinhuang granules, a well-known combined herbal remedy prepared from the extract mixtures of Flos Lonicerae and Radix Scutellariae. The fast HPLC analysis was performed on an Agilent ZorBax SB-C(18) column (4.6×50 mm, 1.8 μm) and 0.2% aqueous formic acid and acetonitrile was the optimum mobile phase for gradient elution in 17 min, which is five times faster than the performance of conventional columns packed with 5.0 μm particles. With various fragmentor voltages in TOF/MS, accurate mass measurements (<5 ppm error) for molecular ions and characteristic fragment ions represented reliable identification criteria for different constituents. A total of 28 compounds, including nine phenolic acids, three iridoid glycosides and nine saponins from Flos Lonicerae and seven flavonoids from Radix Scutellariae, were identified or tentatively characterized in the extract of Yinhuang granules. The established fast HPLC-DAD-TOF/MS method turns out to be useful and efficient for quality control of this commonly used Chinese herbal preparation.     

Separation of natural product using columns packed with Fused‐Core particles

Three HPLC columns packed with 3 μm, sub‐2 μm, and 2.7 μm Fused‐Core (superficially porous) particles were compared in separation performance using two natural product mixtures containing 15 structurally related components. The Ascentis Express^TM C18 column packed with Fused‐Core particles showed an 18% increase in column efficiency (theoretical plates), a 76% increase in plate number per meter, a 65% enhancement in separation speed and a 19% increase in back pressure compared to the Atlantis T3^TM C18 column packed with 3 μm particles. Column lot‐to‐lot variability for critical pairs in the natural product mixture was observed with both columns, with the Atlantis T3 column exhibiting a higher degree of variability. The Ascentis Express column was also compared with the Acquity^TM BEH column packed with sub‐2 μm particles. Although the peak efficiencies obtained by the Ascentis Express column were only about 74% of those obtained by the Acquity BEH column, the 50% lower back pressure and comparable separation speed allowed high‐efficiency and high‐speed separation to be performed using conventional HPLC instrumentation.     

A highly sensitive LC-MS/MS method capable of simultaneously quantitating celiprolol and atenolol in human plasma for a cassette cold-microdosing study

A highly sensitive simultaneous quantitative method for a cassette cold-microdosing study on celiprolol and atenolol was developed with liquid chromatography-tandem mass spectrometry. The method utilizes a combination of solid-phase extraction (SPE) with strong cation exchange (SCX) cartridge columns and reversed-phase chromatography with an ODS analytical column. SCX-SPE cartridge columns (100 mg sorbent) were used for a selective extraction of celiprolol, atenolol and metoprolol (internal standard) from 500 μL of human plasma samples. Turbo-ion spray at positive mode was employed for the ionization of the drug compounds. Quantitation was performed on a triple quadrupole mass spectrometer by selected reaction monitoring with the transitions of m/z 380 to m/z 251 for celiprolol and m/z 267 to m/z 145 for atenolol. Separation of analytes was achieved on an ODS column (100 mm length × 2.1 mm id, 3 μm) by a gradient elution with 10 mM formic acid and methanol by varying their proportion at a flow rate of 0.2 mL/min. The method was validated in the range of 1-250 pg/mL for celiprolol and 2.5-250 pg/mL for atenolol and was successfully applied to the elucidation of pharmacokinetic profiling in a cold cassette microdosing study of the β-blockers.     

The chromatographic interaction and separation of metal ions with 8-Quinolinol stationary phases in several aqueous eluents

Several 8-quinolinol silica gel (QSG) columns were used, with metal-uptake capacities of 10–156 μmol g^?1. Various transition and heavy metal ions were used as analytes in nitrate, sulfate, phosphate, citrate, tartrate, oxalate, phthalate, and maleate mobile phases. Metal-ion retention increased with column capacity and pH. Optimum capacity factors were obtained on columns of intermediate capacity (27 and 46 μmol g^?1). Retention times decreased with an increase in eluent buffer concentration, typically by half with a doubling of buffer. Evidence is presented for the occurence of mobile-phase complexation of analyte ions by eluent buffer species. Multiple or split peaks were often observed when the analyte solvent differed from the mobile phase. Chromatographic separation of up to six metals on the QSG columns is demonstrated in tartrate and maleate mobile phases.     

Practical comparison of LC columns packed with different superficially porous particles for the separation of small molecules and medium size natural products

Commercial C(18) columns packed with superficially porous particles of different sizes and shell thicknesses (Ascentis Express, Kinetex, and Poroshell 120) or sub-2-μm totally porous particles (Acquity BEH) were systematically compared using a small molecule mixture and a complex natural product mixture as text probes. Significant efficiency loss was observed on 2.1-mm id columns even with a low dispersion ultra-high pressure liquid chromatography system. The Kinetex 4.6-mm id column packed with 2.6-μm particles exhibited the best overall efficiency for small molecule separations and the Poroshell 120 column showed better performance for mid-size natural product analytes. The Kinetex 2.1-mm id column packed with 1.7-μm particles did not deliver the expected performance and the possible reasons besides extra column effect have been proved to be frictional heating effect and poor column packing quality. Different column retentivities and selectivities have been observed on the four C(18) columns of different brands for the natural product separation. Column batch-to-batch variability that has been previously observed on the Ascentis Express column was also observed on the Kinetex and Poroshell 120 column.     

Ion chromatography on carbon clad zirconia modified by diazonium chemistry and functionalized latex particles

This work explores the potential of 3 μm carbon coated zirconia particles as a stationary phase for ion chromatography for the separation of organic acids and inorganic ions. A 4-phenylsulfonic acid functionality is introduced onto the carbon surface by reducing 4-phenylsulfonic acid diazonium chloride with borohydride in the presence of carbon clad zirconia particles. The elemental sulfur analysis gave 132 μeq-SO(3)H/g carbon clad zirconia and 2% S atomic concentration by XPS analysis. The -SO(3)(-) groups serve as electrostatic anchors for latex nanoparticles bearing quaternary triethylamine functional groups. The agglomeration step in 5 × 0.4 cm i.d. columns converts the packed particles into an anion exchanger. The breakthrough curves with nitrate indicate a capacity of 3 μeq/column. Separation of common organic acids and inorganic ions using carbonate eluent and suppressed conductivity detection yield plate heights (H) of 0.023-0.05 mm.