Column Outlet Pressure Research Articles

Kinetic mechanism of water dewetting from hydrophobic stationary phases utilized in liquid chromatography

An experimental protocol was designed to accurately measure the dewetting kinetics of aqueous mobile phases from reversed-phase liquid chromatography (RPLC) columns. The protocol enables the determination of the losses in the wetted surface area and internal pore volume (leading to undesirable retention losses) of RPLC columns as a function of the dewetting time. It is used to evaluate the impact of the buffer/salt concentration in water (0–100 mM), nitrogen concentration dissolved in water (0–6.7 × 10−4 M), column temperature (300–358 K), and of the internal structure (pore connectivity) of the stationary phase on the dewetting kinetics of various RPLC packing materials.From a fundamental viewpoint, the experimental facts demonstrate that dewetting kinetics are not solely driven by the pore size of the stationary phase and the contact angle with water. Temperature has a major influence on dewetting kinetics as it controls the nucleation rate of isolated water vapor bubbles over the entire mesoporous network. Additionally, the internal microstructure of the stationary phase (characterized by its internal porosity, pore size distribution, and pore connectivity) influences the rate at which the water vapor bubbles grow and coalesce in the entire particle volume. From a more practical viewpoint, the retention loss of RPLC columns due to water dewetting can be eliminated or at least minimized by (1) adjusting the surface and bonding chemistries to reduce the receding contact angle, (2) elevating the column outlet pressure, (3) operating at the lowest possible temperature, (4) minimizing the pore connectivity of the stationary phase (e.g., by increasing the degree of surface functionalization from C8 to C18-bonded phases), and (5) by degassing the aqueous mobile phase from any dissolved gases.

Instrumental Idiosyncrasies Affecting the Performance of Ultrafast Chiral and Achiral Sub/Supercritical Fluid Chromatography.

It is widely accepted that column technology is ahead of existing chromatographic instruments. The chromatographic output may not reflect the true picture of the peak profile inside the column. The instrumental optimization parameters become far more important when peaks elute in a few seconds. In this work, the low viscosity advantage of the supercritical/subcritical CO2 is coupled with the high efficiency of narrow particle size distribution silica. Using short efficient columns and high flow rates (up to 19 mL/min), separations on the order of a few seconds are demonstrated. In the domain of ultrafast supercritical fluid chromatography (SFC), unexpected results are seen which are absent in ultrafast liquid chromatography. These effects arise due to the compressible nature of the mobile phase and detector idiosyncrasies to eliminate back-pressure regulator noise. We demonstrate unusual connection tubing effects with 50, 75, 127, 254, and 500 μm tubings and show the complex relation of dead time, retention time, efficiency, and optimum velocity with the tubing diameter (via column outlet pressure). Fourier analysis at different back-pressure regulator (BPR) settings shows that some instruments have very specific noise frequencies originating from the BPR, and those specific frequencies vanish under certain conditions. The performance of embedded digital filters, namely, moving average, numerically simulated low pass RC, and Gaussian kernels, is compared. This work also demonstrates, using a simple derivative test, that some instruments employ interpolation techniques while sampling at "true" low frequencies to avoid picking up high frequency noise. Researchers engaged in ultrafast chromatography need to be aware of the instrumental nuances and optimization procedures for achieving ultrafast chiral or achiral separations in SFC mode.

Erratum for: Supercritical Fluid Chromatography as an Alternative Tool for the Qualitative and Quantitative Analysis of Metarhizium brunneum Metabolites from Culture Broth

A fast and selective ultrahigh-performance supercritical fluid chromatography photodiode array detector method was established for the qualitative and quantitative analysis of destruxins, cyclic hexadepsipeptides, from fungal culture broth samples. Prior to analysis, sample purification was carried out using an off-line solid-phase extraction protocol on a reversed-phase material in order to remove unwanted matrix constituents. For separation, detection, and identification, an ultrahigh-performance supercritical fluid chromatography photodiode array detector system hyphenated to a triple quadrupole mass spectrometer was utilized. Analyses were performed on an Acquity ethylene bridged hybrid 2-ethylpyridine sub 2 µm particle size column with CO2 and an acidified (0.02 % trifluor acetic acid) modifier mixture of methanol/acetonitrile (8/2 v/v) serving as mobile phase. For the optimal separation of destruxins, the amount of the modifier was increased in a 10 min linear gradient from 2 % to 20 %, and the column outlet pressure and temperature was set at 140 bars and 60 °C, respectively. Seventeen analytes were separated within an elution window of 4 minutes. Five destruxin congeners (destruxin A, destruxin B, destruxin D, destruxin E, and destruxin E-diol) were identified using reference material. Additionally, eight analytes were tentatively assigned as known destruxins by the evaluation of mass spectrometry data performed as multiple reaction monitoring experiments in the positive electrospray ionization mode.

Supercritical Fluid Chromatography as an Alternative Tool for the Qualitative and Quantitative Analysis of Metarhizium brunneum Metabolites from Culture Broth

A fast and selective ultrahigh-performance supercritical fluid chromatography photodiode array detector method was established for the qualitative and quantitative analysis of destruxins, cyclic hexadepsipeptides, from fungal culture broth samples. Prior to analysis, sample purification was carried out using an off-line solid-phase extraction protocol on a reversed-phase material in order to remove unwanted matrix constituents. For separation, detection, and identification, an ultrahigh-performance supercritical fluid chromatography photodiode array detector system hyphenated to a triple quadrupole mass spectrometer was utilized. Analyses were performed on an Acquity ethylene bridged hybrid 2-ethylpyridine sub 2 µm particle size column with CO2 and an acidified (0.02% trifluor acetic acid) modifier mixture of methanol/acetonitrile (8/2 v/v) serving as mobile phase. For the optimal separation of destruxins, the amount of the modifier was increased in a 10 min linear gradient from 2% to 20%, and the column outlet pressure and temperature was set at 140 bars and 60 °C, respectively. Seventeen analytes were separated within an elution window of 4 minutes. Five destruxin congeners (destruxin A, destruxin B, destruxin D, destruxin E, and destruxin E-diol) were identified using reference material. Additionally, eight analytes were tentatively assigned as known destruxins by the evaluation of mass spectrometry data performed as multiple reaction monitoring experiments in the positive electrospray ionization mode.

Breakthrough Model for Adsorption of Paraffinic Mixtures in Mixed Phase Conditions

A model is developed that allows simulation of breakthrough adsorption experiments of n-alkanes dissolved in a short chain alkane solvent. The model is used to verify conclusions made in previous experimental work that studied competitive adsorption behavior of paraffinic mixtures in vapor and liquid phase conditions. In these types of breakthrough experiments, a pressure drop in the adsorption column results in a change in aggregation state and density changes inside the column, leading to a change in adsorption behavior. Simulations demonstrate the effect of column temperature, outlet pressure, and pellet size on competitive adsorption behavior, and the importance of the effect of density on adsorption behavior is also demonstrated.

Rapid, Direct Quantitation of the Preservatives Benzoic and Sorbic Acid (and Salts) Plus Caffeine in Foods and Aqueous Beverages Using Supercritical Fluid Chromatography

The preservatives benzoate and sorbate, plus caffeine were rapidly separated and quantified, in just over 2 min, in a wide range of beverages and foods, using supercritical fluid chromatography (SFC). Fifteen beverages and 10 semi-liquid foods were evaluated. The benzoate and sorbate were originally present in the samples as the acid, or the sodium, or potassium salt. The aqueous samples were diluted 3:1 with acidified methanol, to insure the acids were protonated, then directly injected. The solutes were isocratically eluted from a 4 × 250 mm, 5 μm Diol column with 3.5 mL min−1 of 8.5 % methanol containing 0.3 % acetic acid at 50 °C and a column outlet pressure of 150 bar. The real samples exhibited remarkably little interference. All the beverages were accurately labeled. However, many of the foods, such as salad dressings, mustard, etc., were mislabeled. The method was linear over a wide range with correlation coefficients for all three solutes >0.999. RSD’s were generally less than 1 %. The results agreed with the caffeine content on the labels within a few percent. Surprisingly, this appears to be the first published separation of benzoic and sorbic acid preservatives in food, and beverages using SFC, and one of a very few SFC applications where aqueous samples were simply diluted and injected. Compared to published references, the SFC method was found to be up to 7 times faster than HPLC, and eliminated the use of acetonitrile.

Use of the isopycnic plots in designing operations of supercritical fluid chromatography. V. Pressure and density drops using mixtures of carbon dioxide and methanol as the mobile phase

The drops of pressure and density along chromatographic columns of different characteristics, eluted with different mixtures of carbon dioxide and methanol was mapped as functions of the column outlet pressure and the operating temperature. This paper extends an earlier report reporting the extent of the pressure and density drops along chromatographic columns eluted with neat CO2[1]. It illustrates the similarities and differences in the pressure and density profiles along columns operated with mixed mobile phases and with neat CO2. Numerical calculations of the pressure and density drops along columns packed with particles of different sizes, under different operating conditions (temperature, outlet pressure, and flow rate), provide important insights regarding the extent of the pressure and density drops under these operating conditions.

A portable, high-speed, vacuum-outlet GC vapor analyzer employing air as carrier gas and surface acoustic wave detection.

Vacuum-outlet GC with atmospheric-pressure air as the carrier gas is implemented at outlet pressures up to 0.8 atm using a low-dead-volume polymer-coated surface acoustic wave (SAW) detector. Increases in the system outlet pressure from 0.1 to 0.8 atm lead to proportional increases in detector sensitivity and significant increases in column efficiency. The latter effect arises from the fact that optimal carrier gas velocities are lower in air than in more conventional carrier gases such as helium or hydrogen due to the smaller binary diffusion coefficients of vapors in air. A 12-m-long, 0.25-mm-i.d. tandem column ensemble consisting of 4.5-m dimethyl polysiloxane and 7.5-m trifluoropropylmethyl polysiloxane operated at an outlet pressure of 0.5 atm provides up to 4 x 10(4) theoretical plates and a peak capacity of 65 (resolution, 1.5) for a 3-min isothermal analysis. At 30 degrees C, mixtures of vapors ranging in vapor pressure from 8.6 to 76 Torr are separated in this time frame. The SAW detector cell has an internal volume of < 2 microL, which allows the use of higher column outlet pressures with minimal dead time. The sensor response is linear with solute mass over at least 2-3 decades and provides detection limits of 20-50 ng for the vapors tested. The combination of atmospheric-pressure air as carrier gas, modest operating pressures, and SAW sensor detection is well-suited for field instrumentation since it eliminates the need for support gases, permits smaller, low-power pumps to be used, and provides sensitivity to a wide range of vapor analytes.

Linear programming of inlet pressure or flow-rate in isothermal gas chromatography with near-vacuum outlet pressure

The equation of motion for a peak in a gas chromatography experiment is shown to be exact whenever the column outlet pressure is approximately zero. Based on steady-state assumptions, this equation is solved for an isothermal analysis with a single-ramp inlet-pressure or flow-rate program. Theoretical calculations of retention times are confirmed experimentally for two n-alkanes using a capillary column connected to a mass-selective detector. A slight but consistent discrepancy between theoretical and measured results is interpreted as a failure of the steady-state assumption when the magnitude of the rate of change of inlet pressure is large.

High-Speed, Vacuum-Outlet GC Using Atmospheric-Pressure Air as Carrier Gas

A model is developed to explore operating conditions and performance tradeoffs for high-speed GC using atmospheric-pressure air as carrier gas and a vacuum pump to draw the carrier gas and injected samples through the system. The model is based on the rate theory for open tubular columns and conventional equations for gas flow in capillary tubes. The model predicts the effects of column outlet pressure, column length, column diameter, and detector dead time on the number of theoretical plates generated in a 30-s analysis spanning a retention factor range from 0 to 5. The outlet (detector) pressure range considered is 1−100 kPa (0.01−1.0 atm). A 0.1-mm-i.d. column is found to generate more plates than either larger or smaller diameter columns because of the constraint of using atmospheric pressure at the column inlet. About 25 000 plates are generated with a 2.5-m-long column for outlet pressures less than ∼20 kPa. The model is validated with a high-speed GC instrument using a cryofocusing inlet system and...

Method development for the separation of phospholipids by subcritical fluid chromatography

A packed-column subcritical fluid chromatographic separation of five phospholipids was achieved in less than 20 min by using a Luna octyl column (25 cm×4.6 mm I.D., 5 μm) employing evaporative light scattering detection. The chromatographic conditions used for the separation of (a) 1,2-dipalmitoyl- sn-glycero-3-phosphate (DPPA), 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), (c) poly(ethylene glycolated)-1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPE–PEG), (d) 1,2-dicaproyl- sn-glycero-3-phosphocholine (DCPC) and (e) 1,2-dipalmitoyl- sn-glycero-3-[phospho- rac-(1-glycerol)] (DPPG) were: 9% ethanol–methanol (50:50, v/v), containing 0.10% (v/v) trifluoroacetic acid for 2 min, increasing by 0.5%/min to 15% in 14 min, to 40% at 14.1 min, at a column temperature of 70°C, an outlet pressure of 135 bar and a mobile phase flow-rate of 2.0 ml/min. The separation was shown to be significantly influenced by the presence of a mobile phase acidic additive (e.g. trifluoroacetic acid) and its concentration, the overall modifier ramp rate and the column outlet pressure.

Pressure-regulating fluid interface and phase behavior considerations in the coupling of packed-column supercritical fluid chromatography with low-pressure detectors

Packed-column supercritical fluid chromatography is usually performed with flow control on the upstream side of the column and pressure control on the downstream side. This arrangement is compatible with detectors that can be operated at column-outlet pressure and placed between the column outlet and a pressure-regulating device. However, mass transfer problems may arise when low-pressure detectors like a mass spectrometer or evaporative-light-scattering detector are used with downstream pressure regulators or programmable nozzles. These problems can be avoided by replacing the regulator or nozzle with a tee delivering a pressure-regulating make-up fluid from a pressure-controlled pump. Parameters are chosen to avoid the liquid–vapor two-phase regions of the phase diagram for the resulting mixture. We demonstrate this approach and show how to use phase diagrams and their pressure–temperature projections for setting operating conditions.

Supercritical fluid chromatograph for studies of retention and efficiency with a brief study of n-alkanes showing evidence of significant temperature drop with pressure drop

An instrument designed specifically to examine retention and efficiency in packed-column supercritical fluid chromatography is described. Built on a gas chromatograph, it incorporates a syringe pump, helium-actuated injector, flame ionization detector, two outlet restrictors, and direct control of column inlet and outlet pressures. A supercritical fluid solvent injection system provides chromatograms with no solvent peak or solvent-induced effects on retention and efficiency. Mass flow-rates obtained by reading the volumetric displacement at the syringe pump are accurate to within 1.4%, and the repeatability of retention times and plate numbers with the pump operated in constant pressure and constant flow modes is examined. Isopycnic plate height curves for elution of n-dodecane, n-tetradecane, n-hexadecane, and n-octadecane with carbon dioxide at 50°C and a reduced density of 1.0 on a 150×2.0-mm I.D. column packed with 10-μm Spherisorb C 8 show a broad minimum with reduced plate heights of about 2 at reduced velocities from 3 to 7, with sharp increases at higher velocities for the heavier solutes. Retention volumes decrease with pressure drop, which is attributed to a significant temperature drop from the inlet to the outlet of the column.

Chiral separations by packed-column super- and subcritical fluid chromatography

The higher diffusivity and lower viscosity of supercritical and near-critical fluids can make packed-column supercritical fluid chromatography or subcritical fluid chromatography (SubFC) faster with improved resolution over normal-phase HPLC for chiral separations. However, superior fluid characteristics do not guarantee enhanced resolution or shorter analysis time. In the case of phenylalaninol, a 1H NMR spectrum indicates interaction of the solute with carbon dioxide. Such interaction may explain the poorer resolution achieved by SubFC than by HPLC. For a secondary amine, its 1H NMR spectra showed no shift while poorer resolution was again observed for SubFC when compared to HPLC. Thus, the NMR data by itself do not conclusively indicate either reaction between basic solutes and carbon dioxide or the likelihood of chiral resolution. The effects of column outlet pressure, organic modifier composition, pump flow-rate and column temperature for hydroxyzine were studied. Of the physical parameters studied, modifier composition has the greatest impact on retention. Increasing retention generally increases resolution. Changing temperature generally has less impact on retention but produces the greatest selectivity changes.

Analysis of triazine and triazole herbicides by gradient-elution supercritical fluid chromatography

A mixture of substituted triazine and triazole herbicdes is separated on an analytical-scale column by gradient elution using supercritical fluid CO 2 with increasing methanol content and flow-rate. High efficiency and fast analysis times are achieved with complete resolution of all the compounds. The effect of CO 2 flow-rate on the separation has been studied and it is shown that an increase in the overall mobile phase flow-rate by a factor of two reduces the analysis time drastically with no loss in resolution. Also, the effects of the oven temperature and the column outlet pressure on the separation have been studied.

New pressure regulating system for constant mass flow supercritical-fluid chromatography and physico-chemical analysis of mass-flow reduction in pressure programming by analogous circuit model

The newly developed regulating valve is based on high-speed flow switching, and is capable of controlling the column outlet pressure independent of the mass flow-rate of the fluid. The reproducibility of retention times in pressure-programming elution on a 4.6 mm i.d. ×250mm long silica gel column was better than 1% with supercritical carbon dioxide as the mobile phase. The outlet mass flow-rate decreased while the pressure was increasing, keeping the input flow to the system constant; this was due to the compressibility of the fluid. The amount of decrease in the outlet flow was calculated by introducing an analogous circuit model and new parameters, instrumental and column capacitances. The result agreed with the measured value within 30% error.

The effect of column characteristics on the minimum analyte concentration and the minimum detectable amount in capillary gas chromatography

AbstractThe need for faster and more efficient separations of complex mixtures of organic compounds by gas chromatography has led to the development of small inner diameter open tubular columns. Owing to their decreased plate height, extremely narrow peaks are obtained. When differently sized columns with equal plate numbers are compared, injection of a fixed amount of a solute will give the highest detector signals for the smallest bore columns. When P is defined as the ratio of the column inlet and outlet pressures, it can be seen from theory that under normalized chromatographic conditions the minimum detectable amount (Qº) for a mass flow sensitive detector increases proportionally to the square of the column diameter for P = 1. In the situation of greater interest in the practice of open tubular gas chromatography where P is large, a linear relationship is derived between Qº and the column diameter.It is a widespread misunderstanding, however, that narrow bore capillary columns should be used for this reason in trace analysis. If a fixed relative contribution of the injection band width to the overall peak variance is allowed, a decreased plate height drastically restricts the maximum sample volume to be injected. It is shown that the minimum analyte concentration in the injected sample (Cº) is inversely proportional to the column inner diameter when a mass flow sensitive detector is used. For actual concentrations less than Cº, sample preconcentration is required. The effect of peak resolution and selectivity of the stationary phase in relation to Cº and Qº will be discussed as well. The validity of the given theory is experimentally investigated. Minimum analyte concentrations and minimum detectable amounts are compared using columns with different inner diameter.

Optimum performance of capillary GC columns as a function of tube diameter and film thickness under various operating conditions. Computer program for calculation of H‐ū curves and minimum analysis times

AbstractThe Golay‐Giddings and Poiseuille equations are used to derive equations for the calculation of the maximum plate number and minimum time conditions for given columns at fixed, but selectable, outlet pressures. In addition, expressions are presented for the determination of minimum analysis times for separation problems requiring given plate numbers. In this instance, the optimum column length can be calculated as a function of outlet pressure. A Basic computer program, incorporating the equations for the various optima, together with the H‐ū curves, is described. Input variables are either column length or desired plate number, column diameter, film thickness, capacity ratio of the solute, column outlet pressure, seperation temperature, and carrier gas. The carrier gas viscosity is automatically calculated in the case of hydrogen, helium, or nitrogen. For these gases, and if the solute is a n‐alkane, the diffusivity of the solute in the mobile phase is calculated. In this case, the carbon number of the solute is needed in the computation.For high molecular weight polydimethylsilicone phases (e.g. SE‐30), the program can approximate the diffusivity of n‐alkanes in the stationary phase at the given temperature as a function of the carbon number. Of course, manually entered values of viscosity and diffusion coefficients can be included in the calculations.

Model describing the role of the pressure gradient on efficiency and speed of analysis in capillary gas chromatography

Neglecting the term describing resistance to mass transfer in the stationary phase, the Golay plate height equation is rearranged in terms of the dimensionless parameters ξ = H/H min and ν = u / ̄ ū opt. In the resulting model, two boundary cases can be distinguished: P ≈ 1 and P ≫ 1, P being the ratio of column inlet to column outlet pressure. The two expressions provide a clear insight in the increase in plate height which results from the use of average linear carrier gas velocities other than u opt. Hence the model is very helpful for optimization of the speed of analysis. The validity of both boundary expressions was checked by comparing them with experimental plate height data. The data under conditions of P ≫ 1 were obtained on a 60 m × 0.4 mm I.D. capillary column directly coupled to the ion source of a mass spectrometer, and on a 10 m x 55 μm I.D. capillary column operated at increased inlet pressure, using atmospheric outlet pressure. A 30 m × 0.4 mm I.D. column was tested using a flame ionization detector, with P ≈ 1. Excellent agreement was observed between the theoretical prediction and the experimental results.

Gas Chromatography under the Column Outlet Pressure Higher than Atmospheric

Abstract The effects of column outlet pressures on the HETP vs. carrier gas velocity curves are discussed on the basis of KOS theory. It is shown that when a column is operated at a constant outlet pressure higher than atmospheric the minimum of H-\baru curve shifts towards lower HETP and \baru values, and on the contrary the operation at reduced outlet pressure results in the shift of the minimum towards higher HETP and \baru values. The separation factor and number of theoretical plates obtained by a column operated at the outlet pressure of 4.0 atm are superior by ca. 10% to those obtained by normal gas chromatography.