pH-zone-refining counter-current chromatography: Origin,mechanism, procedure and applications

Since 1980, high-speed counter-current chromatography (HSCCC) has been used for separation and purification of natural and synthetic products in a standard elution mode. In 1991, a novel elution mode called pH-zone refining CCC was introduced from an incidental discovery that an organic acid in the sample solution formed the sharp peak of an acid analyte. The cause of this sharp peak formation was found to be bromoacetic acid present in the sample solution which formed a sharp trailing border to trap the acidic analyte. Further studies on the separation of DNP-amino acids with three spacer acids in the stationary phase revealed that increased sample size resulted in the formation of fused rectangular peaks, each preserving high purity and zone pH with sharp boundaries. The mechanism of this phenomenon was found to be the formation of a sharp trailing border of an acid (retainer) in the column which moves at a lower rate than that of the mobile phase. In order to facilitate the application of the method, a new method was devised using a set of retainer and eluter to form a sharp retainer rear border which moves through the column at a desired rate regardless of the composition of the two-phase solvent system. This was achieved by adding the retainer in the stationary phase and the eluter in the mobile phase at a given molar ratio. Using this new method the hydrodynamics of pH-zone-refining CCC was diagrammatically illustrated by three acidic samples. In this review paper, typical pH-zone-refining CCC separations were presented, including affinity separations with a ligand and a separation of a racemic mixture using a chiral selector in the stationary phase. Major characteristics of pH-zone-refining CCC over conventional HSCCC are as follows: the sample loading capacity is increased over 10 times; fractions are highly concentrated near saturation level; yield is improved by increasing the sample size; minute charged compounds are concentrated and detected at the peak boundaries; and elution peaks are monitored with a pH flow meter for compounds with no chromophore. Since 1994, over 70 research papers on pH-zone-refining CCC have been published with the trends increasing in the recent years.     

The efficient analysis of neutral and highly polar pharmaceutical compounds using reversed-phase and ion-exchange electrochromatography

Summary Highly polar compounds, such as tricyclic antidepressants are very difficult to analyse by electrochromatography with conventional reversed-phase silica-based chromatography packings. At high pH (high electroosmotic flow) the test compounds were not eluted from a Spherisorb ODS-1 column, as a result of strong interactions between the analyte and residual silanol groups on the packing material. By lowering the pH of the mobile phase, whereby the highly basic test compounds become positively charged, it was possible to elute the samples but only with severe peak tailing. Because the electroosmotic flow was greatly reduced, the elution time for neutral species became prohibitively long. By use of a strong cation exchanger in place of C₁₈-silica it was found possible to resolve a series of highly basic compounds with very high efficiencies, with very little evidence of peak tailing. Plate numbers in excess of 8 million per metre were observed.     

Gradient elution in counter-current chromatography: a new layout for an old path

Gradient elution in CCC is a powerful tool, which needs further systematic development to become robust and easy to use. The first attempt to build a correlation between gradient elution profile and distribution ratio (K(D)) values for model mixtures containing typical representatives of pharmaceutical compounds is presented in this paper. The three step estimation of the solvent system composition of a heptane-ethyl acetate-methanol-water (HEMWat) series is described. The estimation is based on simple measurements of initial and final stationary phase retention for gradient elution run, calculating gradient distribution ratio and correlating it with static K(D) against HEMWat number.     

Characterization of carboxylic acids in atmospheric aerosols using hydrophilic interaction liquid chromatography tandem mass spectrometry

A sensitive hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry (HILIC/ESI-MS/MS) method was developed for determination of selected aliphatic (i.e. malonic, succinic, glutaric, adipic, pimelic, suberic, azelaic, maleic, fumaric, glycolic and pyruvic acid), alicyclic (i.e. cis-pinonic and pinic acid) and aromatic (i.e. trimesic, phthalic acid and its isomers) carboxylic acids. Analytes were separated on an amide column using a gradient elution with a 10mM constant ionic strength mobile phase containing acetonitrile and aqueous ammonium acetate buffer (pH 5.0). The influence of the buffer type, pH, polar modifier and temperature on analyte retention under HILIC was studied. Static sonication-assisted solvent extraction was optimized for sample preparation prior to analysis. The recoveries obtained were higher than 90% for most analytes. The method was proven to be sensitive with limits of detection ranged from 0.03 to 16.0 μg/L in selected reaction monitoring mode (SRM). The repeatability and intermediate precision of the method, expressed as RSD (%) of the peak area ratio between analytes and their internal standards were generally lower than 5%. The method was successfully applied for determination of the studied acids in samples of ambient aerosol particles. A big advantage of the new method is also its ability to detect and separate the isobaric compounds of the selected carboxylic acids. Our results demonstrate that the method is specific and sensitive for the determination of a wider range of polar carboxylic acids at low concentrations in complex samples of aerosol particles.     

Band splitting in overloaded isocratic elution chromatography I. The experimental evidence

A series of samples of increasing volume (from 0.001 to 4.0 cm3), containing the same constant concentration (40 g/l) of two simple compounds, ethylbenzoate and 4-tert.-butylphenol were injected on a Kromasil-C18 column with methanol-water (62:38. v/v) as the mobile phase. Complex band profiles were observed when the volume of the sample became large enough and strong band interference took place. The analysis of the fractions collected during the elution of the mixed band demonstrates that, for samples larger than 2 cm3, the band of 4-tert.-butylphenol is split into two separate bands, one eluted before and the other eluted after the band of ethylbenzoate. Such a phenomenon has never been observed yet in RPLC, under isocratic elution conditions.     

High-performance liquid chromatography of selenium compounds utilizing perfluorinated carboxylic acid ion-pairing agents and inductively coupled plasma and electrospray ionization mass spectrometric detection

Increasing speciation demands in clinical chemistry, toxicology and nutrition have made the determination of the total elements in a sample inadequate; the amount of an element and the chemical forms in which it is present need to be known. Inductively coupled plasma mass spectrometry (ICP-MS) was used after high-performance liquid chromatographic (HPLC) separation, as was electrospray ionization mass spectrometry (ESI-MS). The effect of variation of the number of carbon atoms in perfluorinated carboxylic acids used as ion-pairing agents for the separation of selenium compounds was examined. Trifluoroacetic acid (0.1%), pentafluoropropanoic acid (0.1%) or heptafluorobutanoic acid (0.1%; HFBA) were alternatively used as additives to methanol-water (1:99, v/v) solutions as mobile phases. Reversed-phase HPLC-ICP-MS with 0.1% HFBA in the mobile phase allowed more than 20 selenium compounds to be separated in 70 min in an isocratic elution mode; the separation of natural selenium-enriched sample extracts was examined and explained. The pH of the 0.1% HFBA solution was modified with hydrochloric acid or ammonia and the pH of the sample extracts before injection was modified in order to overcome unwanted double peak formation in the chromatograms of sample extracts. Oxidations of standard gamma-glutamyl-Se-methylselenocysteine and Se-methylselenocysteine were carried out using 30% H2O2 solution and identifications of selenium-containing oxidation products were made using HPLC-ICP-MS and HPLC-ESI-MS. The principal organic oxidation product in both cases was methaneseleninic acid (MeSeO2H).     

Reverse Phase High Performance Liquid Chromatography of Human Bence Jones Proteins

Abstract

A high performance liquid chromatography system is presented for analytical and preparative separations of human Bence Jones proteins. The method utilizes 5–7 um macroreticular polystylene resin with bonded hydroxymethyl functional groups, and the proteins are eluted with a linear gradient of an increasing concentration of acetonitorile(10–60%, V/V) in 0.1 % (V/V) trifluoroacetic acid, pH 2.1. By this elution condition, seven X type Bence Jones proteins with molecular weights of 23,600 (monomer)-47,000(dimers) daltons(216–434 amino acids) were eluted within 80 min with the yields of 78%–98%. The method allows a rapid and sharp elution of Bence Jones proteins.     

反相高效液相色谱法分离重组促红细胞生成素

利用反相高效液相色谱法对重组促红细胞生成素进行了纯化。结果表明,利用Ｃ４反相柱和乙腈－三氟乙酸流动相在洗脱梯度和上样样品纯度等条件都较适当时,可以简单、快速、高效地从粗品中分离出重组促红细胞生成素,获得的产品纯度高,接近１００％;比活性好,约为１．９６×１０８ＩＵ／ｇ蛋白。     

Dual-mode high-speed counter-current chromatography: retention, resolution and examples

Counter-current chromatography (CCC) is a very versatile technique offering high resolution power in recovering very pure compounds from complex matrices. Dual-mode CCC where the phase role is reversed during the separation is investigated here; it ensures elution of all the injected species from the column while, unlike backflush, the separation is still progressing after phase reversal; equations giving retention and retention factor are derived from the basic equations of chromatography. Compared to normal-mode CCC, it is shown that enhanced resolution in dual-mode CCC can be obtained in conditions derived from a theoretical model. The experimental section provides the validation of the retention prediction while resolution is also proven to be enhanced in dual-mode CCC. However, equations given in the theoretical section cannot fully explain the results obtained for resolution because they do not deal with kinetics. Dual-mode CCC has also been applied to separation of polyoxypropylene glycol polymers; separation can be achieved with a small number of experiments because all the injected compounds are eluted by reversing the phases. Dual-mode CCC also gave improved yields in the purification of antibiotics compared with previous results using normal-mode CCC.     

Ion-pair liquid chromatography--atmospheric pressure ionization mass spectrometry for the determination of quaternary ammonium herbicides

High-performance liquid chromatography coupled with atmospheric pressure ionization mass spectrometry (electrospray and atmospheric pressure chemical ionization) has been used to characterize some quaternary ammonium herbicides (quats). The separation of these compounds was carried out using ion-pair chromatography with heptafluorobutyric acid (15 mM, pH 3.3) and acetonitrile gradient elution for successful coupling to mass spectrometry. Detection limits down to 0.1-4 micrograms l-1 were obtained for spiked tap water following a preconcentration step. Good reproducibilities (day-to-day and run-to-run) were also obtained.     

High-performance liquid chromatographic separation of bilirubin conjugates: the effects of change in molarity and pH

A fast, sensitive high-performance liquid chromatographic method has been developed for the separation and quantitation of biliary bile pigments; this utilizes a C18 reversed-phase column with two solvents, a buffer and an organic solvent, which were changed in a linear gradient from a polar to a less polar combination. Nine glycosidic conjugates of bilirubin as well as unconjugated bilirubin and a suitable internal standard, unconjugated mesobilirubin IX alpha, were all separated to baseline by gradient elution; the species eluted in a polar to less polar fashion. Increasing the molarity of the solvent decreased the binding of non-glucuronide pigments to the column, with a decrease in their retention times, whereas for bilirubin monoglucuronide they increased. Decrease in pH, similarly, preferentially increased bilirubin monoglucuronide retention times.     

Resolution of three forms of superoxide dismutase by immobilised metal affinity chromatography.

A new method for separation of three forms of superoxide dismutase (SOD) using immobilised metal affinity chromatography (IMAC) is reported. Fe-, Mn- and Cu/Zn-SODs were eluted sequentially from Cu(2+)-IMAC column with an increasing gradient of a counter ion (NH+4) run in combination with an increasing pH gradient (6.8-7.8). The combined gradient elution method resulted in separation of SODs with high resolution, the three proteins being eluted in electrophoretically homogeneous forms. Similar preparation could not be achieved by either increasing gradient of a counter ion or decreasing pH gradients used separately. The described methodology has been successfully applied for separation of three SODs from a protozoan parasite, indicating that this combined gradient elution system for IMAC offers new possibilities for the high-resolution separation of proteins exhibiting only minor differences in their amino acid composition and structure.     

Changes in the order of elution of some organic compounds in high-resolution gas chromatography on polar columns depending on the injection method and water content in the sample

During the GC analysis of apple aroma essence using a capillary column with polar stationary phases, i.e., bonded/crosslinked polyethylene glycol (HP-Innowax) and acid-modified polyethylene glycol (FFAP) the phenomenon of changing the elution order of some compounds was observed ("cross-over phenomenon"). The elution order within two pairs of closely eluting compounds: 2-methylbutyl acetate/pentan-3-ol (internal standard) and trans-2-hexen-1-al/2-methylbutan-1-ol differed between samples introduced onto the column with direct injection and with static headspace method. Experiments carried out on standard solutions of investigated compounds in water-acetone mixtures of gradually reduced water ratio (99:1 to 0:100) proved that changes of elution times leading first to co-elution and subsequently to reversion of elution order of compounds within both pairs depended on the amount of water in the injected sample. The same effect was observed while the amount of water was diminished by reducing the sample volume introduced into the column.     

Efficient Separation of Phytochemicals from Muehlenbeckia volcanica (Benth.) Endl. by Polarity-Stepwise Elution Counter-Current Chromatography and Their Antioxidant,Antiglycation, and Aldose Reductase Inhibition Potentials

Muehlenbeckia volcanica (Benth.) Endl. (M. volcanica), native to South America, is a traditional Peruvian medicinal plant that has multi-therapeutic properties; however, no phytochemicals have been identified from it yet. In this study, a five-step polarity-stepwise elution counter-current chromatography (CCC) was developed using methanol/water (1:5, v/v) as the stationary phase and different ratios of n-hexane, ethyl acetate, and n-butanol as mobile phases to separate the compounds from the 70% methanol extract of M. volcanica, by which six compounds with a wide range of polarities were separated in a single run of CCC and were identified as gallic acid, protocatechuic acid, 4,4′-dihydroxy-3,3′-imino-di-benzoic acid, rutin, quercitrin, and quercetin. Then, two compounds from the fractions of stepwise elution CCC were separated using conventional high-speed CCC, pH-zone-refining CCC, and preparative high-performance liquid chromatography, and identified as shikimic acid and miquelianin. These compounds are reported from M. volcanica for the first time. Notably, except for shikimic acid, all other compounds showed anti-diabetic potentials via antioxidant, antiglycation, and aldose reductase inhibition. The results suggest that the polarity-stepwise elution CCC can be used to efficiently separate or fractionate compounds with a wide range of polarities from natural products. Moreover, M. volcanica and its bioactive compounds are potent anti-diabetic agents.     

Measuring Peak Capacity of Reversed-Phase Columns for Small Molecule Compounds Under Gradient Elution

The conventional approach to the measurement of peak capacity of reserved-phase columns under gradient elution assumes that all peaks have constant peak width, but this assumption can lead to inaccurate measurement of peak capacity. An integration approach employing a series of alkylphenones as model compounds was employed to more accurately measure the peak capacity for small molecule compounds under gradient elution. The base peak width of alkylphenones was plotted against retention time and a peak width function over retention time was established by polynomial regression. The peak capacity was then calculated by integrating the inverse of the peak width function over a gradient window. Compared to the conventional method, the integration method is not based on the assumption of equal peak width, thus providing a more accurate measurement of the peak capacity of reserved-phase columns, especially shorter ones packed with sub-2 μm particles under gradient elution.     

Elution behavior of polyethylene in polar mobile phases on a non-polar sorbent

Linear polyethylene standards in the range of 1-500 kg/mol, dissolved in 1,2,4-trichlorobenzene, were injected into a column packed with oligo(dimethylsiloxane) modified silica gel. Fifteen polar solvents (cyclohexanone, cyclohexylacetate, cyclohexanol, nonylalcohol, dimethylformamide, dimethyl sulfoxide, ethylene- and diethylene glycol monobutyl ether, benzylalcohol, hexylacetate, bis(2-ethyl-hexyl)phthalate, N,N-dimethylacetamide, propylene carbonate, dipropylene glycol and N-methyl-pyrrolidone) were evaluated as mobile phases. Depending on the type of mobile phase evaluated, different elution behaviors are observed for polyethylene: (1) polyethylene was eluted in the size exclusion mode, (2) polyethylene was eluted together with the sample solvent peak at constant elution volume, (3) polyethylene was partially or fully retained on the column. The retained polymer was easily removed from the column by injecting a small volume of trichlorobenzene. The use of ethylene glycol monobutyl ether as the mobile phase enabled separation of the polyethylene from polypropylene. In this case polypropylene is eluted in the size exclusion mode, while polyethylene is eluted at a constant elution volume or remains in the column.     

Direct extraction of urinary analytes from undeveloped reversed-phase thin layer chromatography plates using a solvent gradient combined with on-line electrospray ionisation ion mobility-mass spectrometry

The direct extraction of urinary analytes deposited on reversed-phase thin-layer chromatography (RP-TLC) plates is demonstrated using a solvent gradient extraction procedure without prior chromatographic development. The surface sample probe TLC-MS interface used for the gradient extraction is compared to direct loop injection into the electrospray ion source for biofluid profiling. The gradient elution is shown to enhance ion intensities, as urinary salts are eluted in aqueous formic acid in the early part of the gradient reducing ion suppression. The retention of urinary components on the C18 RP-TLC plate was confirmed by monitoring analyte responses with, and without, an aqueous wash phase prior to the solvent gradient extraction. The use of gradient elution allows fractionation of the complex biological matrix as a result of differential retention of urine components on the undeveloped RP-TLC plate. The direct gradient analysis of TLC plates has also been combined with ion mobility-mass spectrometry to further resolve the complex urinary profile and identify co-eluting compounds.     

液质联用分析葛根提取物及中药片剂中异黄酮类化合物

采用反相C18毛细管液相色谱柱,以乙腈(含0.1%(体积分数,下同)三氟乙酸)和水(含0.1%三氟乙酸)为流动相梯度洗脱,在26 min内分离了葛根异黄酮提取物以及愈风宁心片中的主要成分。采用毛细管液相色谱/四极杆飞行时间串联质谱仪对葛根提取物以及片剂中的几种主要异黄酮类化合物做了结构分析,发现葛根素是主成分(提取物中其平均质量分数是13.32%;片剂中每片含量19.28~24.34 mg)。对微量未知化合物,用它们的子离子谱图与已知化合物的谱图比较,推测其成分为3′-甲氧基葛根素和3′-甲氧基大豆苷。     

Detection of low-abundance impurities in synthetic thyroid hormones by stationary phase optimized liquid chromatography–mass spectrometry

The transfer of a gradient method to an isocratic or multistep gradient method employing stationary phase optimized liquid chromatography facilitated a reduction in analysis time by 50% and significantly improved the mass spectrometric detectability of impurities in synthetic thyroid hormones. Four column segments packed with different stationary phases were combined into a single chromatographic column, which allowed the separation and photometric as well as mass spectrometric detection of thyroid compounds in less than 30 min under isocratic- or step gradient elution conditions with 0.10% acetic acid/acetonitrile. Signal instability and baseline drift during detection by negative electrospray ionization time-of-flight mass spectrometry were minimized by optimizing the spray parameters for each individual elution step. This resulted in improved detectabilities and higher mass spectral quality, especially for low-abundance components in the sample mixture. The method was applied to the separation and detection of the low-abundance impurities formed upon the thermal stressing of a sample of synthetic levothyroxine.     

Sample conditions to avoid pH distortion in RP‐LC

Band deformations might take place for acids and bases in preparative applications and adsorption studies where it is necessary to use overloaded injections. In this study, we focus on how deformations can be prevented without using highly concentrated buffers that may precipitate in the eluent. We have systematically investigated how the elution zones depend on which protolytic form the analyte has when it is dissolved. Basic and acidic model compounds are investigated using eluents with different pH values and the resulting elution profiles are compared when the analytes are dissolved in their protonated and deprotonated form, i.e., in uncharged form or as different kinds of salts. Depending on the analyte's protolytic form, a sample zone is created at the column inlet whose pH deviates, more or less, from the bulk eluent's. If the local adsorption strength in this sample zone is greater than the bulk eluent's, the elution profiles are compressed. Under opposite conditions, the eluted bands are more or less deformed and may even be split; completely different deformations can even take place for different kinds of salt combinations. Explanations of these, and other, effects, together with detailed guidelines for proper sample preparation to avoid peak deformations, are given.