Characterization and classification of stationary phases in HPLC and SFC – a review

Packed column supercritical fluid chromatography (pSFC) is an attractive technique in drug discovery related analysis because it offers several advantages over the more commonly used high-performance liquid chromatography (HPLC) technique. The environmental-friendly CO₂ mobile phase, the high-throughput capacity, the increased efficiency and the lower operational costs give SFC additional benefits over HPLC in analysis related to drug development. The latter technique is well established and has been used for decades in the pharmaceutical industry. On the other hand, SFC is still in its infancy, even though the technique has been known for decades and researchers are still discovering the possibilities and limitations of this technique.     

Comparative study of new shell-type,sub-2 μm fully porous and monolith stationary phases,focusing on mass-transfer resistance

Today sub-2 μm packed columns are very popular to conduct fast chromatographic separations. The mass-transfer resistance depends on the particle size but some practical limits exist not to reach the theoretically expected plate height and mass-transfer resistance. Another approach applies particles with shortened diffusion path to enhance the efficiency of separations. In this study a systematical evaluation of the possibilities of the separations obtained with 5 cm long narrow bore columns packed with new 2.6 μm shell particles (1.9 μm nonporous core surrounded by a 0.35 μm porous shell, Kinetex™, Core-Shell), packed with other shell-type particles (Ascentis Express™, Fused-Core), totally porous sub-2 μm particles and a 5 cm long narrow bore monolith column is presented. The different commercially available columns were compared by using van Deemter, Knox and kinetic plots. Theoretical Poppe plots were constructed for each column to compare their kinetic performance. Data are presented on polar neutral real-life analytes. Comparison of a low molecular weight compounds (MW = 270–430) and a high molecular weight one (MW ∼ 900) was conducted. This study proves that the Kinetex column packed with 2.6 μm shell particles is worthy of rivaling to sub-2 μm columns and other commercially available shell-type packings (Ascentis Express or Halo), both for small and large molecule separation. The Kinetex column offers a very flat C term. Utilizing this feature, high flow rates can be applied to accomplish very fast separations without significant loss in efficiency.     

Investigating sub-2 μm particle stationary phase supercritical fluid chromatography coupled to mass spectrometry for chemical profiling of chamomile extracts

Roman and German chamomile are widely used throughout the world. Chamomiles contain a wide variety of active constituents including sesquiterpene lactones. Various extraction techniques were performed on these two types of chamomile. A packed-column supercritical fluid chromatography–mass spectrometry method was designed for the identification of sesquiterpenes and other constituents from chamomile extracts with no derivatization step prior to analysis. Mass spectrometry detection was achieved by using electrospray ionization. All of the compounds of interest were separated within 15 min. The chamomile extracts were analyzed and compared for similarities and distinct differences. Multivariate statistical analysis including principal component analysis and orthogonal partial least squares-discriminant analysis (OPLS-DA) were used to differentiate between the chamomile samples. German chamomile samples confirmed the presence of cis- and trans-tonghaosu, chrysosplenols, apigenin diglucoside whereas Roman chamomile samples confirmed the presence of apigenin, nobilin, 1,10-epioxynobilin, and hydroxyisonobilin.     

Stage-frit: A straightforward sub-2 μm nano-liquid chromatography column fabrication for proteomic analysis

In this work we demonstrated a facile method for the fabrication of C18 coordination polymer gel in a capillary, called stage-frit, which was efficiently applied to pack sub-2 μm C18 beads into the capillary by a high pressure bomb for the online separation of proteolytic peptides. The back pressure of the column with 10 cm × 75 μm i.d. is regularly lower than 170 bar at a flow rate of 300 nl/min, which could be operated on a common nanoLC system instead of nanoUPLC system due to the good permeability, low back pressure and high mechanical stress of the frit that will totally reduce the cost for the purchase of instrument. The stage-frit allows long-term continuous flow of the solvent and no significant beads loss or pressure instability was observed during the period. The repeatability of retention time for fifteen BSA tryptic peaks was found to be less than 1.08% (RSD) in six time nanoLC-ESI-MS/MS experiments. The average full width at half maximum (FWHM) of peptide peaks is 5.87 s. The sub-2 μm stage-frit nanoLC column showed better sensitivity than the commercial available for large scale proteomic analysis of total tissue proteins from human spleen. The number of identified peptides is approximately 0.4-fold and 0.2-fold higher than that obtained by utilizing commercial columns packed with 3 μm and 1.8 μm C18 materials, respectively. In the field of analytical chemistry, particularly the use of nanoLC systems, stage-frit nanoLC column offers a great potential for the separation of complex mixtures.     

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.     

Synthesis and chromatographic evaluation of new polymeric chiral stationary phases based on three (1<Emphasis Type="Italic">S</Emphasis>,2<Emphasis Type="Italic">S</Emphasis>)-(−)-1,2-diphenylethylenediamine derivatives in HPLC and SFC

Three new polymeric chiral stationary phases were synthesized based on (1S,2S)-1,2-bis(2,4,6-trimethylphenyl)ethylenediamine, (1S,2S)-1,2-bis(2-chlorophenyl)ethylenediamine, and (1S,2S)-1,2-di-1-naphthylethylenediamine via a simple free-radical-initiated polymerization in solution. These monomers are structurally related to (1S,2S)-1,2-diphenylethylenediamine which is the chiral monomer used for the commercial P-CAP-DP polymeric chiral stationary phase (CSP). The performance of these three new chiral stationary phases were evaluated in normal phase high-performance liquid chromatography (HPLC) and supercritical fluid chromatography and the results were compared with those of the P-CAP-DP column. All three new phases showed enantioselectivity for a large number of racemates with a variety of functional groups, including amines, amides, alcohols, amino acids, esters, imines, thiols, and sulfoxides. In normal phase, 68 compounds were separated with 28 baseline separations (Rs ≥ 1.5) and in SFC, 65 compounds were separated with 24 baseline separations. In total 72 out of 100 racemates were separated by these CSPs with 37 baseline separations. Complimentary separation capabilities were observed for many analytes. The new polymeric CSPs showed similar or better enantioselectivities compared with the commercial column in both HPLC and SFC. However, faster separations were achieved on the new stationary phases. Also, it was shown that these polymeric stationary phases have good sample loading capacities while maintaining enantioselectivity.     

Modern analytical supercritical fluid chromatography using columns packed with sub-2 μm particles: A tutorial

This tutorial provides an overview of the possibilities, limitations and analytical conditions of modern analytical supercritical fluid chromatography (SFC) using columns packed with sub-2 μm particles. In particular, it gives a detailed overview of commercially available modern SFC instrumentation and the detectors that can be employed (UV, MS, ELSD, FID, etc.). Some advice on the choice of the stationary phase dimensions and chemistries, the nature of the mobile phase (choice of organic modifier and additives) and its flow rate as well as the backpressure and temperature are also provided. Finally, several groups of potentially problematic compounds, including lipophilic compounds, hydrophilic substances and basic drugs, are discussed in detail. All these families of analytes can be resolved with SFC but require specific analytical conditions.     

Fast gradient screening of pharmaceuticals with 5 cm long, narrow bore reversed-phase columns packed with sub-3 μm core-shell and sub-2 μm totally porous particles

The performance of 5 cm long narrow-bore columns packed with 2.6-2.7 μm core-shell particles and a column packed with 1.7 μm totally porous particles was compared in very fast gradient separations of polar neutral active pharmaceutical compounds. Peak capacities as a function of flow-rate and gradient time were measured. Peak capacities around 160-170 could be achieved within 25 min with these 5 cm long columns. The highest peak capacity was obtained with the Kinetex column however it was found that as the flow-rate increases, the peak capacity of the new Poroshell-120 column is getting closer to that obtained with the Kinetex column. Considering the column permeability, peak capacity per unit time and per unit pressure was also calculated. In this comparison the advantage of sub-3 μm core-shell particles is more significant compared to sub-2 μm totally porous particles. Moreover it was found that the very similar sized (d_p = 2.7 μm) and structured (ρ = 0.63) new Poroshell-120 and the earlier introduced Ascentis Express particles showed different efficiency. Results obtained showed that the 5 cm long narrow bore columns packed with sub-3 μm core-shell particles offer the chance of very fast and efficient gradient separations, thus these columns can be applied for fast screening measurements of routine pharmaceutical analysis such as cleaning validation.     

High temperature-high efficiency liquid chromatography using sub-2 μm coupled columns for the analysis of selected non-steroidal anti-inflammatory drugs and veterinary antibiotics in environmental samples

A high efficiency HPLC method was developed by coupling three sub-2 μm columns in series and operating them at high temperature for the separation of selected non-steroidal anti-inflammatory drugs and veterinary antibiotics in environmental samples. The separation was performed at 80 °C to reduce the solvent viscosity, thus reducing the column backpressure. The chromatographic performance of high temperature-extended column length HPLC method was used to determine the most widely used non-steroidal anti-inflammatory drugs and veterinary antibiotics such as sulphonamides in wastewater samples. The method could simultaneously determine 24 pharmaceuticals in short analysis time with high efficiency. The method involved pre-concentration and clean-up by solid phase extraction (SPE) using Oasis HLB extraction cartridges. It was validated based on linearity, precision, detection and quantification limits, selectivity and accuracy. Good recoveries were obtained for all analytes ranging from 72.7% to 98.2% with standard deviations not higher than 6%, except for acetaminophen and acetyl salicylic acid, for which low recovery was obtained. The detection limits of the studied pharmaceuticals ranged from 2 to 16 μg L⁻¹, while limits of quantification were in the range from 7 to 54 μg L⁻¹ with UV detection.     

Metabolic profiling of amino acids in cellular samples via zwitterionic sub-2 μm particle size HILIC-MS/MS and a uniformly 13C labeled internal standard

A novel analytical method using hydrophilic interaction liquid chromatography combined with electrospray tandem mass spectrometry for metabolic profiling of free, underivatized amino acids is presented. The separation uses a zwitterionic modified silica-based stationary phase with 1.8-μm particle size functionalized with ammonium sulfonic acid groups. Quantification is based on external standard calibration using a Pichia pastoris cell extract grown on uniformly ¹³C labeled glucose as an internal standard. The absolute limits of detection in the cellular matrix were in the subpicomolar range. Measurement accuracy was assessed by analyzing NIST Standard Reference Material 2389a, which provides certified values for 17 amino acids. The recovery of the amino acids ranged between 65 % (proline) and 120 % (lysine), with excellent repeatability precision below 2.5 % (n?=?5). Only, cystine showed poor recovery (29 %) and repeatability precision (13 %). Generally, the long-term precision obtained by hydrophilic interaction liquid chromatography–tandem mass spectrometry was excellent, being on average less than 9 % over 20 h of measurement time. Moreover, the novel separation method had average repeatability and reproducibility of the chromatographic peak width over time periods of 20 h and 6 months of 8 and 15 %, respectively, demonstrating its high robustness in routine analysis of cellular samples. Large concentration differences depending on the amino acid were found in the cell extracts, typically ranging from 0.002 nmol per milligram of cell dry weight (cystine) to 56 nmol per milligram of cell dry weight (arginine and glutamic acid).     

Thermal stability and 2.7 μm spectroscopic properties in Er3+ doped tellurite glasses

In present paper, the thermal stability and 2.7 μm spectroscopic properties in Er³⁺ doped tellurite glasses have been investigated by 980 nm laser diode pumping. Thermal analysis indicates that GeO₂ modified tellurite glass has better thermal stability and anti-crystallization ability. Judd-Ofelt intensity parameters are calculated and discussed to examine the covalency characteristics based on absorption spectra. The 2.7 μm fluorescence is obtained and the lifetime can reach 124 ± 1 μs with the quantum efficiency of 61.5% in prepared samples. Moreover, higher effective emission bandwidth (136.67 nm), emission cross sections (12.75 × 10⁻²¹ cm²) and radiative transition probability (95.66s⁻¹) at 2.7 μm are achieved. In addition, upconversion and near-infrared emission spectra are measured to elucidate energy transfer mechanism of Er³⁺. The results suggest that the present tellurite glass modified by GeO₂ might have promising applications in mid-infrared fiber lasers.     

Analytical and semi-preparative HPLC enantioseparation of novel pyridazin-3(2H)-one derivatives with α-aminophosphonate moiety using immobilized polysaccharide chiral stationary phases

The direct HPLC enantioseparation of a novel series of chiral pyridazin-3(2H)-one derivatives with α-aminophosphonate moiety was performed on two immobilized polysaccharide chiral stationary phases (Chiralpak IA, Chiralpak IC) using n-hexane (n-Hex)/dichloromethane (DCM) mobile phase with 5% alcohol additive. Good baseline separation of the enantiomers was achieved using amylose tris-(3,5-dimethylphenylcarbamate) chiral stationary phases (Chiralpak IA) on analytical scale. The analytical method was further scaled up to semi-preparative loading to obtain small amounts of both the enantiomers of pyridazin-3(2H)-one derivative. The semi-preparative resolution of all compounds was successfully achieved with n-hexane/dichloromethane/ethanol (EtOH) as mobile phase using a semi-preparative Chiralpak IA column. The first fractions were isolated with purities of >99.9% (enantiomeric excess (e.e.), and the second fractions were obtained with purities of >98.2% (enantiomeric excess). The assignment of the absolute configuration was established for the F1 fraction of compound a-2 by single-crystal X-ray diffraction method.     

Rapid high performance liquid chromatography method development with high prediction accuracy,using 5 cm long narrow bore columns packed with sub-2 μm particles and Design Space computer modeling

Many different strategies of reversed phase high performance liquid chromatographic (RP-HPLC) method development are used today. This paper describes a strategy for the systematic development of ultrahigh-pressure liquid chromatographic (UHPLC or UPLC) methods using 5 cm × 2.1 mm columns packed with sub-2 μm particles and computer simulation (DryLab^® package). Data for the accuracy of computer modeling in the Design Space under ultrahigh-pressure conditions are reported. An acceptable accuracy for these predictions of the computer models is presented. This work illustrates a method development strategy, focusing on time reduction up to a factor 3–5, compared to the conventional HPLC method development and exhibits parts of the Design Space elaboration as requested by the FDA and ICH Q8R1. Furthermore this paper demonstrates the accuracy of retention time prediction at elevated pressure (enhanced flow-rate) and shows that the computer-assisted simulation can be applied with sufficient precision for UHPLC applications (p > 400 bar). Examples of fast and effective method development in pharmaceutical analysis, both for gradient and isocratic separations are presented.     

Standardized particle size for monitoring, inventory, and assessment of metals and other trace elements in sediments: <20 microm or <2 microm?

Despite the manifold factors that determine the composition of a river sediment (e.g., geology, soil type, rural or highly industrialized and densely populated, as well as forested areas, concentration of carbonates and organic matter), the linear regressions between the 9 metals determined in the fractions <2 microm and <20 microm have high coefficients (R2 after Pearson) for Cr and Cu (both 0.94), followed by Pb (0.90), Cd (0.82), Zn (0.81), Ni (0.76), and Mn (0.72). Low and very low coefficients are found for Hg (0.51) and Fe (0.22). In addition, the histograms of the metal ratios <2 microm:<20 microm (i.e. the slope of a linear regression) show that--with the exception of Cd (1.47)--all median ratios of the other metals fall within a narrow range (1.24-1.35). Both "fine" (<2 microm and <20 microm) fractions are able to fulfil the requirements for monitoring, inventory, and assessment of metals in sediments. Preference should be given to the rapid, simple, and economic <20 microm separation by sieving; this fraction corresponds fairly closely to the former suspended load of a riverine transport.     

Critical comparison of performances of superficially porous particles and sub-2 μm particles under optimized ultra-high pressure conditions

The performance of 2.7 μm superficially porous particles at 600 bar and sub-2 μm fully porous particles at 1000 bar were compared by the Poppe plot method. Theoretical Poppe plots were first constructed for each stationary phase to compare their kinetic performance at different analysis times. The theory was then verified by experiments under the optimized conditions identified from the Poppe plot calculation. We found that the 2.7 μm superficially porous particles at 600 bar can provide similar performance compared to the sub-2 μm fully porous particles at ultra-high pressure (1000 bar) when analysis times are very short (e.g. sub-minute). As analysis time increases, the superficially porous particles start to outperform the sub-2 μm particles and can give much higher efficiencies (e.g. > 2 times higher plate count) at very long analysis times (>3 h). The comparison was extended to gradient elution of a mixture of pharmaceutical interest by constructing gradient peak capacity Poppe plots and similar behavior was observed.     

Preparation and chiral recognitio of a novel chiral stationary phase for HPLC,based on mono(6~A-N-1-(2-hydroxyl)-phenylethylimino-6~A-deoxy)-β-cyclodextrin and covalently bonded silica gel

A novel chiral stationary phase (CSP)was prepared by immobilizing mono(6~A-N-1-(2-hydroxyl)-phenylethylimino-6~A- deoxy)-β-cyclodextrin onto the surface of silica gel via a longer spacer.This chiral stationary phase exhibited good enantios- electivity for a variety of chiral compounds under reversed-phase conditions.     

Preparation and chiral recognition of a novel chiral stationary phase for HPLC,based on mono(6A-N-1-(2-hydroxyl)-phenylethylimino-6A-deoxy)-β-cyclodextrin and covalently bonded silica gel

A novel chiral stationary phase (CSP) was prepared by immobilizing mono(6A-N-1-(2-hydroxyl)-phenylethylimino-6A-deoxy)-β-cyclodextrin onto the surface of silica gel via a longer spacer.This chiral stationary phase exhibited good enantios electivity for a variety of chiral compounds under reversed-phase conditions.     

Preparation and chiral recognition of a novel chiral stationary phase for HPLC,based on mono（6A-N- 1-（2-hydroxyl）- phenylethylimino-6A-deoxy）-β-cyclodextrin and covalently bonded silica gel

A novel chiral stationary phase （CSP） was prepared by immobilizing mono（6A-N-1-（2-hydroxyl）-phenylethylimino-6A- deoxy）-β-cyclodextrin onto the surface of silica gel via a longer spacer. This chiral stationary phase exhibited good enantioselectivity for a variety of chiral compounds under reversed-phase conditions.