High-performance Liquid Chromatography Column Research Articles

Column Watch: Methods for the Passivation of HPLC Instruments and Columns

Metal-analyte interaction is a type of nonspecific adsorption (NSA) that occurs mainly between acidic analytes and active adsorptive sites present in the metal surfaces of the fluidic path of any chromatographic system. NSA represents a chromatographic challenge since it may lead to poor peak shape, low recovery, and poor performance from the high performance liquid chromatography (HPLC) system. As a result, many solutions that circumvent and mitigate this type of secondary interaction have been published or commercialized in the last few years. Such solutions encompass swapping stainless steel components for non-reactive polymers, “biocompatible” metals or alloys, the addition of additives into the mobile phase, pretreating the system with large amounts of sample or strong acids, and the use of coated stainless steel materials. This article explores these solutions in more detail, particularly the recent adaptation and benefits of coated metal surfaces in LC.

Determination of Related Substances in Promethazine Hydrochloride and Dextromethorphan Hydrobromide in Oral Solution by RP-HPLC Method.

The goal of this study is to provide a single, widely applicable high-performance liquid chromatographic (HPLC) technique for the determination of related substances in multicomponent oral solution of promethazine hydrochloride and dextromethorphan hydrobromide. For the assessment of impurities of promethazine hydrochloride and dextromethorphan hydrobromide in oral solution, a unique, sensitive, quick, stability-indicating gradient HPLC technique has been created. For chromatographic separation, an Agilent Eclipse XDB-C18, 250mm × 4.6mm, 5μm column was used with a buffered mobile phase consisting of a mixture of potassium dihydrogen phosphate pH3.0:acetonitrile (80:20) v/v as mobile phase A and potassium dihydrogen phosphate pH3.0:acetonitrile:methanol (10:10:80) v/v/v as mobile phase B. The separation was performed at a flow rate of 1.2mL/min and a detection wavelength of 224nm. The temperature of the column oven was regulated at 40°C. With good sensitivity and resolution, all compounds were effectively separated on a reverse-phase HPLC column. Acid, base, photolytic, thermal, oxidative and humidity stress conditions significantly degraded dextromethorphan hydrobromide and promethazine hydrochloride. The developed technique was validated according to the criteria of the International Conference on Harmonization for all validation parameters such as specificity, accuracy, linearity, precision, the limit of detection, the limit of quantitation and robustness.

Reducing pressure and increasing peak capacity by incorporating an adaptable flow stream splitting platform to high-performance liquid chromatography columns: A study on C18 silica-based monoliths.

In this study, an adaptable end-column platform was fitted to a commercially available monolith, which enabled the column to be fitted with a flow-splitting device. A variety of flow-splitting adapters could be incorporated into the platform, and in this study, a radial flow stream splitter was utilized. The advantage of the radial flow stream spitter was that it overcame issues relating bed density variations that could cause bands to distort in the radial cross-section of the column. Using propylbenzene as a test standard in isocratic elution mode, height equivalent to a theoretical plate curves were constructed across ten flow rates, and it was found that the column efficiency improved by as much as 73%. Furthermore, the dual outlet flow splitter enabled a very substantial reduction in column back pressure, with the decrease being consistently between 20 to 30% depending on the column length. Additionally, sensitivity increased by 45%, consistent with the observed increase in efficiency. The adaptable end-column platform could be retrofitted to almost any commercial column with the expectation of gaining efficiency, sensitivity, and reducing back pressure.

Effect-Directed Profiling of Akebia quinata and Clitoria ternatea via High-Performance Thin-Layer Chromatography, Planar Assays and High-Resolution Mass Spectrometry.

Two herbal plants, Akebia quinata D. leaf/fruit and Clitoria ternatea L. flower, well-known in traditional medicine systems, were investigated using a non-target effect-directed profiling. High-performance thin-layer chromatography (HPTLC) was combined with 11 different effect-directed assays, including two multiplex bioassays, for assessing their bioactivity. Individual active zones were heart-cut eluted for separation via an orthogonal high-performance liquid chromatography column to heated electrospray ionization high-resolution mass spectrometry (HPLC-HESI-HRMS) for tentative assignment of molecular formulas according to literature data. The obtained effect-directed profiles provided information on 2,2-diphenyl-1-picrylhydrazyl scavenging, antibacterial (against Bacillus subtilis and Aliivibrio fischeri), enzyme inhibition (tyrosinase, α-amylase, β-glucuronidase, butyrylcholinesterase, and acetylcholinesterase), endocrine (agonists and antagonists), and genotoxic (SOS-Umu-C) activities. The main bioactive compound zones in A. quinata leaf were tentatively assigned to be syringin, vanilloloside, salidroside, α-hederin, cuneataside E, botulin, and oleanolic acid, while salidroside and quinatic acids were tentatively identified in the fruit. Taraxerol, kaempherol-3-rutinoside, kaempferol-3-glucoside, quercetin-3-rutinoside, and octadecenoic acid were tentatively found in the C. ternatea flower. This straightforward hyphenated technique made it possible to correlate the biological properties of the herbs with possible compounds. The meaningful bioactivity profiles contribute to a better understanding of the effects and to more efficient food control and food safety.

High formation of trichloroacetic acid from high molecular weight and ultra-hydrophilic components in freshwater raphidophytes upon chlorination

Raphidophytes are flagellate unicellular algae that causes algal blooms in drinking water sources. In Japan, it was recently reported that the concentration of trichloroacetic acid (TCAA), a major chlorinated disinfection byproduct (DBP), increased dramatically in drinking water when the source water contained raphidophytes. Additionally, raphidophytes produced haloacetic acid (HAA) precursors, especially TCAA precursors, in high concentrations. However, their properties are still unknown, and thus, well-designed countermeasures against DBP formation have not yet been established. Therefore, in this study, the HAA precursors originated from raphidophytes in natural water collected from the algal blooms in Muro Dam (Nara Prefecture, Japan) and Gonyostomum semen (G. semen), a raphidophyte species, cultivated in the laboratory, were characterized to provide the information for establishing suitable treatment strategies. Using several high-performance liquid chromatography columns, solid-phase extraction cartridges, and ultrafiltration devices, and the spectral profiles, we discovered that the HAA precursors are highly hydrophilic and high-molecular-weight compounds with acidic and phenolic functional groups. Further characterization of the high-molecular-weight fraction (> 3 kDa) from the G. semen culture showed that the HAA precursors had a molecular weight of ~10–60 kDa, and that they were not protein molecules despite containing a large amount of nitrogen atoms. Furthermore, the TCAAFP of the fraction (310 ± 25 μg/mg C) were as high as phenol, known as a reactive TCAA model precursor. The presence of unique and unreported DBP precursors was confirmed.

Separation of chiral compounds using high performance liquid chromatography stationary phase based on covalent organic framework material TpPa-NH2-Glu

Covalent organic frameworks （COFs） are an emerging class of porous crystalline materials composed of multidentate organic units connected by covalent bonds. COFs have been demonstrated to exhibit great potential and research value in many fields， including gas storage and separation， photoelectric devices， fluorescence sensors， catalysis， drug delivery， dye and pollutant adsorption， and electronic devices， and so on. The COFs obtained by post-synthesis modification tend to exhibit high crystallinities and porosities， thereby rendering them suitable materials for use in the fields of chiral resolution， asymmetric catalysis， and chromatography. In this work， TpPa-NO2 was synthesized from 1，3，5-tricarbaldehyde phloroglucinol and 2-nitro-1，4-phenylenediamine， which was then reduced to TpPa-NH2. Subsequently， this material was modified with D-glucose via a post-synthesis modification strategy to obtain the TpPa-NH2-Glu. TpPa-NH2-Glu were characterized by nuclear magnetic resonance （NMR） spectroscopy， Fourier transform-infrared （FT-IR） spectroscopy， X-ray powder diffraction （XRD） analysis， etc. In the XRD pattern， the peaks observed at 4.7°， 8.1°， 11.1°， and 27° were attributed to the TpPa-NH2-Glu， and these peaks are consistent with previous reports， thereby confirming the successful synthesis of this derivative. In addition， circular dichroism experiments indicated that the TpPa-NH2-Glu exhibited a Cotton effect， further confirming the chiral COF was prepared. Subsequently， this material was immobilized on the surface of spherical silica gel particles via the net-wrapping method to prepare a stationary phase for high performance liquid chromatographic column. Using n-hexane-isopropanol （9∶1， v/v） or methanol-water （9∶1， v/v） as mobile phases at a flow rate of 0.5 mL/min， 16 racemates and two benzene-based positional isomers （o，m，p-nitroaniline and o，m，p-Iodoaniline） were successfully resolved by this chiral column. In addition， under methanol-water （9∶1， v/v） mobile phase conditions， five racemates were separated， among which propranolol hydrochloride， warfarin， and metoprolol reached baseline separation. Furthermore， under n-hexane-isopropanol （9∶1， v/v） mobile phase conditions， 11 racemates were resolved， among which ethyl 2-bromopropionate and 3-butyn-2-ol reached baseline separation. Meanwhile， the effect of temperature on the TpPa-NH2-Glu liquid chromatography column and the repeatability of the TpPa-NH2-Glu liquid chromatography column were also explored. The HPLC column prepared by TpPa-NH2-Glu had good repeatability， and its relative standard deviation （RSD） was 1.55% and 1.46%， respectively. It is demonstrated that the TpPa-NH2-Glu material has good resolution ability for chiral compounds.

Direct analysis of tobacco specific nitrosamines in tobacco products using a molecularly imprinted polymer-packed column.

Tobacco specific nitrosamines (TSNAs) are highly carcinogenic by-products in tobacco samples, and their presence is regulated by the Food and Drug Administration. Molecularly imprinted polymers (MIPs) are synthetic polymers that have been "imprinted" with a template analyte in a co-polymer system, and can selectively extract analytes from complex matrices. MIPs can be incorporated into online systems, replacing traditional high performance liquid chromatography (HPLC) columns. MIP material specific for TSNAs was packed into an empty HPLC column using a slurry packing technique. The developed method with the MIP-packed HPLC column was validated on a LC-MS/MS system for the quantitation of N-nitrosonornicotine (NNN) and 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in commercial tobacco products. The method was linear over .1-10 ng/ml (.4-10 μg/g) for NNN and NNK. The limit of detection (LOD) was .03 ng/ml (12 μg/g) and the limit of quantitation (LOQ), .1 ng/ml (.4 μg/g). All column uniformity parameters with the exception of theoretical plate number were within the accepted criteria (% RSD values <15%). Theoretical plate number was <250, owing to the large (50 μm) sized MIP particles. Twenty-six tobacco products contained TSNA concentrations that were consistent with reported literature values. The TSNA-MIP based HPLC column effectively replaced a traditional reverse phase HPLC column, and was used for the direct analysis of nicotine and tobacco products without extensive sample preparation prior to instrumental analysis.

Evaluation of separation performance for eggshell-based reversed-phase HPLC columns by controlling particle size and application in quantitative therapeutic drug monitoring.

Eggshell-based reversed-phase packing materials were applied to an analytical column for high-performance liquid chromatography. Commercially available eggshell powder was classified by a cyclone system to obtain three types of particles with different diameters (arithmetic mean ± standard deviation: 4.3 ± 3.8, 5.6 ± 3.3, and 9.5 ± 5.5 μm). Sedimentation separation removed tiny particles from each sample, resulting in particles with arithmetic means of 6.6 ± 5.5, 7.3 ± 4.5, and 10.2 ± 5.0 μm, respectively. The unclassified particles and three particle types treated with sedimentation separation were subsequently packed into analytical columns (150 mm × 4.6 mm I.D.), and their separation efficiencies were evaluated by comparing their height equivalent to a theoretical plate (HETP). The column without sedimentation separation exhibited the highest HETP, whereas the columns with sedimentation separation showed better separation efficiency and lower back pressure. The column with the best separation efficiency was applied for the separation of 10 alkylbenzenes and 5 steroids, and all peaks were observed with complete separation (peak resolution: RS > 1.5). Finally, the column was used for quantitative analysis of voriconazole, an azole antifungal agent, and imatinib, a first-generation molecularly targeted drug for cancer treatment, in spiked whole blood. Excellent accuracy (99.1-102.8%) and precision (0.6-1.9%) were observed for the spiked drugs and long-term stability (>3000 column volumes of mobile phase flow) indicated good applicability of the developed eggshell-based column as an analytical column for routine analyses of therapeutic drugs in blood.

Studies on the key constituents and the related mechanisms of Clerodendranthus spicatus in the treatment of diabetes based on network pharmacology

Ethnopharmacological relevanceClerodendranthus spicatus is a traditional Chinese medicine and has been used to treat diabetes and some kidney diseases for a long history. Aim of the studyThe research aimed to study the active constituents, the potential targets and the related mechanisms of C. spicatus in the treatment of diabetes through network pharmacology method and verify the antidiabetic activity by molecular biology experiments. Materials and methodsA comprehensive network pharmacology strategy was used to predict the key active constituents, the key targets and the related mechanisms and pathways of C. spicatus in the treatment of diabetes. The strategy mainly included screening and predicting potential active constituents and targets by network construction, GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis. Based on the predicted results, C. spicatus was extracted by ultrasonic method with 50% ethanol and enriched by using macroporous resin. The compounds with potential antidiabetic effects were separated through silica-gel column chromatography and HPLC (high performance liquid chromatography), and then identified by MS (mass spectrum) and NMR (nuclear magnetic resonance). The C. spicatus extract and isolated compounds were tested by in-vitro and cell experiments to verify their antidiabetic activities, including antioxidant activities, inhibition activities on α-glucosidase and α-amylase, the influence on glucose uptake in cell experiments and the Western blot of PI3K and Akt expression levels. ResultsA total of 18 active constituents and 16 key targets of C. spicatus in the treatment of diabetes were screened out through network pharmacology method. Phenolic acids might be the main target compounds for the next research. After extraction, enrichment and separation, the phenolic acids-enriched fraction of C. spicatus and four phenolic acid compounds (helisterculin C, salvianolic acid B, orthosiphoic acid E and ethyl caffeate) were obtained. Among them, salvianolic acid B was isolated from C. spicatus for the first time and orthosiphoic acid E was isolated from natural products for the first time. In experiment verification, the crude extract of C. spicatus, the phenolic acids-enriched fraction and the four compounds all showed antidiabetic potentials. The phenolic acids in C. spicatus had antioxidant activities, inhibitory activities on α-amylase and α-glucosidase and promoted glucose uptake in L6 cells through PI3K/Akt signaling pathway. ConclusionsThis study showed that C. spicatus had antidiabetic activities with the mechanism of the mode of multi-compounds acting on multi-targets and multi-pathways. The main active phenolic acid compounds were also identified. It provided theoretical basis for further development and utilization of C. spicatus.

Selective Separation of Thyroid-Hormone-Receptor-Binding Substances Using Molecularly Imprinted Polymers.

To date, an identification protocol for endocrine disruptors that bind to the thyroid hormone receptor (TR) has not been established. A method for screening and identifying TR-binding substances is highly required due to the existence of unknown TR-binding substances from the environment. Here, we conceived a chromatographic method using a molecularly imprinted polymer (MIP) to create a novel screening protocol for the endocrine disruptors. A receptor-imitating MIP was prepared using N-acetylthyroxine (AcetylT4) and 4-vinylpyridine as a pseudo-template and a functional monomer, respectively, based on the existing molecular recognition mechanism of the TR. The receptor-imitating MIP provided molecular recognition ability for all the TR-binding substances that were employed in this study. The prepared MIPs were packed into a high-performance liquid chromatography column for the simultaneous analysis of TR-binding and non-binding substances. The former was strongly retained, while the latter was not. The presence or absence of TR-binding/non-binding activity resulted in successful dichotomous separation. Additionally, the surface imprinting technique was applied to improve the separation performance of the MIP packing material. MIP-coated uniformly sized silica-based particles of 5 μm were successfully prepared, and the MIP-coated silica column enabled more efficient dichotomous separation of TR-binding and non-binding substances.

“Click” preparation of a chiral macrocycle-based stationary phase for both normal-phase and reversed-phase high performance liquid chromatography enantioseparation

Chiral polyimine macrocycles (CPMs) constitute a new family of organic macrocycles that have defined cavities, rigid shapes, inherent chirality and multiple cooperative binding sites, and have shown great potential in diverse areas. However, the application of CPMs for high performance liquid chromatography (HPLC) enantioseparation has rarely been reported. In this work, a novel chiral stationary phase (CSP) for HPLC was prepared by chemical bonding of a CPM (C54H72N6O3) onto thiolated silica via thiol-ene click reaction. The CSP exhibited good enantioselectivity in both normal- and reversed-phase HPLC. Chiral compounds included alcohols, diols, ketones, organic acids, esters, ethers, amines, and epoxides were enantioseparated on the column in normal-phase mode (17 compounds) and reversed-phase mode (20 compounds). Importantly, broader chiral resolution was observed with the column than that obtained using our previously studied chiral macrocycle H3L-based column, indicating the potential to significantly improve and broaden applicability of this novel macrocycle-type CSPs. Moreover, the CSP exhibited good complementary enantioseparation to Chiralpak AD-H and Chiralcel OD-H columns, enabling separation of some racemates that could not be separated by the two popular chiral HPLC columns. In addition, the fabricated column exhibited good stability and reproducibility. The relative standard deviations (RSDs) (n = 5) of retention time and resolution after multiple injections were < 0.20 % and < 0.39 %, respectively. The results demonstrated the great potential of this type of CPM for HPLC separation of enantiomers.

Preparation of Porous Silica Particles with a Controlled Mesopore Size by Ultrasonic Spray Drying for High-Performance Liquid Chromatography (HPLC)

Highly spherical porous silica particles (PSPs) with a controlled pore size were prepared by ultrasonic spray drying and were used as a packing material for high-performance liquid chromatography (HPLC). By using organic additives added to the silica sol as porogens instead of low-melting salts, choosing the size of the colloidal SiO2 particles, and varying the type and the ratio of additives, the pore size of the PSPs was finely tuned from 2.9 to 34.4 nm. Subsequently, PSPs having pore sizes of 7.4, 13.5, and 29.0 nm were modified by chlorodimethyloctadecylsilane (PSPs-C18), and HPLC columns packed with the different PSPs-C18 were evaluated systematically using aromatic compounds and alkylbenzene derivatives as probes. A plate number of 81,000/m and the relative standard deviation of less than 1% in the retention time were obtained for pentylbenzene over 100 injection and HPLC separation cycles for the 7.4-PSPs-C18 column. Five proteins were also separated within 25 min using the 29.0-PSPs-C18 column.

Separation of the enantiomers of underivatized amino acids by using serially connected dual column high-performance liquid chromatography-tandem mass spectrometry

In this article, a serially connected dual column liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the simultaneous separation and enantioseparation of proteinogenic amino acids. For this purpose, different achiral and chiral stationary phases (CSP) and mobile phase compositions have been tested. As a result of the optimization studies, the best enatioseparation for amino acids were achieved with a combination of zwitterionic and crown ether stationary phases using a gradient of two mobile phases: A (water:TFA 99.5:0.5, % v/v) and B (acetonitrile:ethanol:TFA 85:15:0.5, % v/v/v). The developed method provided simultaneous enantioseparation of all proteinogenic amino acids under this study including isomeric and isobaric ones except for proline. The method was successfully applied to human lung adenocarcinoma cells (A549) and healthy human lung epithelial cells (BEAS-2B) cultivated with d-amino acid containing cocktails in order to evaluate d-amino acids transfer rate in normal and cancer lines. Thed/l amino acid ratios were different in cancer and normal cell lines cultivated as mentioned above for aspartic acid, cysteine, methionine, phenylalanine, and serine.

Rapid determination of 16 polycyclic aromatic hydrocarbons in atmospheric fine particles by molecularly imprinted column-high performance liquid chromatography

A high performance liquid chromatographic method based on the purification of polycyclic aromatic hydrocarbon molecularly imprinted columns was developed, this method was used for the rapid quantitative determination of 16 polycyclic aromatic hydrocarbons(PAHs) in fine particulate matter(PM_(2.5)). The polycyclic aromatic hydrocarbons of the sample were extracted with acetonitrile ultrasonically, purified by molecular imprinting column(MIP-PAHs), and tested after being concentrated by nitrogen blowing. The 16 PAHs showed a good linear relationship in the range of 1-40 ng/mL, and the correlation coefficient r&gt;0.9996; the recoveries were 77.13%-107.67% at the three standard addition levels of 5.0, 10.0 and 20.0 ng/mL, the relative standard deviation was 0.15%-4.63%(n=7); the detection limit of the method was 0.004-0.078 ng/m~3, and the quantification limit was 0.016-0.336 ng/m~3. The detection of 16 PAHs in PM_(2.5) using this method is more accurate, more sensitive and less time consuming than the result of the simple ultrasonic extraction method and the Soxhlet extraction method in Determination of PAHs in ambient air and exhaust gases in gas phase and particulate matter by high performance liquid chromatography(HJ 647-2013).

Preparation and evaluation of a chiral porous organic cage based chiral stationary phase for enantioseparation in high performance liquid chromatography

Porous organic cages (POCs) are a new kind of porous molecular materials, which have gained widespread interest in many fields due to their intriguing properties, including excellent molecular solubility, inherent molecular cavity and rich host-guest chemistry. To date, many chiral POCs have been explored as chiral stationary phases (CSPs) for gas chromatographic (GC) separation of enantiomers. However, the applications of chiral POCs for high performance liquid chromatography (HPLC) enantiomeric separation is extremely rare. In this study, we report the construction of thiol-ene click reaction for the preparation of CSP for HPLC by using a [4+8]-type chiral POC NC4-R as chiral selector. The fabricated CSP showed good chiral resolution performance not only in normal-phase HPLC (NP-HPLC) but also in reversed-phase HPLC (RP-HPLC). Seventeen and ten racemates were well resolved in the two separation modes, respectively, including ketones, esters, alcohols, phenols, amines, ethers, organic acids, and amino acids. Moreover, the fabricated column also shows good chiral recognition complementarity to two popular chiral HPLC columns (Chiralpak AD-H and Chiralcel OD-H columns) and previously reported chiral POC NC1-R-based HPLC column, which can resolve some racemates that unable to be resolved by the two commercially available chiral HPLC columns and NC1-R-based column. The relative standard deviation (RSD) values (n = 4) of retention time and resolution (Rs) of analytes separated on the column were less than 0.3 % and 0.5 % after it was subjected to different injections, showing the good reproducibility and stability of the NC4-R-based column. This work demonstrated high potentials of chiral POCs for HPLC enantioseparation and the applicability of chiral POC-based HPLC columns can be broadened by developing more chiral POCs with diverse structures as chiral selector for HPLC.

Enhancement of Proteome Coverage by Ion Mobility Fractionation Coupled to PASEF on a TIMS-QTOF Instrument.

Trapped ion-mobility spectrometry (TIMS) was used to fractionate ions in the gas phase based on their ion mobility (V s/cm2), followed by parallel accumulation-serial fragmentation (PASEF) using a quadrupole time-of-flight instrument to determine the effect on the depth of proteome coverage. TIMS fractionation (up to four gas-phase fractions) coupled to data-dependent acquisition (DDA)-PASEF resulted in the detection of ∼7000 proteins and over 70,000 peptides overall from 200 ng of human (HeLa) cell lysate per injection using a commercial 25 cm ultra high performance liquid chromatography (UHPLC) column with a 90 min gradient. This result corresponded to ∼19 and 30% increases in protein and peptide identifications, respectively, when compared to a default, single-range TIMS DDA-PASEF analysis. Quantitation precision was not affected by TIMS fractionation as demonstrated by the average and median coefficient of variation values that were less than 4% upon label-free quantitation of technical replicates. TIMS fractionation was utilized to generate a DDA-based spectral library for downstream data-independent acquisition (DIA) analysis of lower sample input using a shorter LC gradient. The TIMS-fractionated library, consisting of over 7600 proteins and 82,000 peptides, enabled the identification of ∼4000 and 6600 proteins from 10 and 200 ng of human (HeLa) cell lysate input, respectively, with a 20 min gradient, single-shot DIA analysis. Data are available in ProteomeXchange: identifier PXD033129.

Modifying the Metal Surfaces in HPLC Systems and Columns to Prevent Analyte Adsorption and Other Deleterious Effects

Interactions of certain analytes with metal surfaces in high performance liquid chromatography (HPLC) instruments and columns cause a range of deleterious effects, including peak broadening and tailing, low peak areas, and the formation of new peaks due to chemical reactions. To mitigate these effects, we have developed a novel surface modification technology in which a hybrid organic/inorganic surface based on an ethylene-bridged siloxane chemistry is applied to the metal components in HPLC instruments and columns. We demonstrate the impact of this technology on peak symmetry, peak area, and injection-to-injection and column-to-column reproducibility for several metal-sensitive analytes. We also show an example of the mitigation of an on-column oxidation reaction. A variant of this technology has recently been developed for size-exclusion chromatography of proteins. An example is shown demonstrating the use of this variant applied to size-exclusion columns for the separation of a monoclonal antibody monomer and higher molecular weight species. Together, these results highlight the importance of preventing interactions of analytes with metal surfaces in HPLC in order to achieve accurate and precise results.

VALIDASI METODE ANALISIS SENYAWA ETIL p-METOKSISINAMAT DALAM PLASMA DARAH TIKUS PUTIH JANTAN GALUR WISTAR MENGGUNAKAN KROMATOGRAFI CAIR KINERJA TINGGI

Ethyl p-methoxycinnamate (EPMC) isolated from the rhizome of kencur (Kaempferia galanga L) is being developed at STFI. Validation of EPMC analysis in blood plasma using High-Performance Liquid Chromatography (HPLC) needs to be conducted to perform in vivo EPMC analysis. This study aims to validate the EPMC analysis method in blood plasma using HPLC. The HPLC column used is column C18, with methanol : phosphoric acid 70:30 and 80:20 mobile phases, a flow rate of 1.0 mL/min, and a maximum wavelength of 310 nm. The blood plasma used was the blood plasma of white male rats of the Wistar strain. EPMC was added to blood plasma and analyzed using HPLC. Parameter validation used is system suitability test, linearity, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ). The results showed that the optimum mobile phase for analyzing EPMS in plasma was methanol : phosphoric acid with a ratio of 70:30. Based on the validation, the linear regression was 0.998. In the accuracy test, the average value range for the percent recovery is 98% - 102%. In the precision test, the average value of the RSD percentage was 4.10% - 68.83%. The LOD value is 2.747 g/mL, and the LOQ value is 9.158 g/mL. Based on this research, it can be concluded that EPMC in blood plasma can be analyzed using KKKT with column C18 and mobile phase methanol : phosphoric acid 70:30.

Types of High-Performance Liquid Chromatography (HPLC) Columns: A Review

Some chemical analysts, especially those working in the food sector, often find it difficult to choose the HPLC columns for their research. Every so often they tend to modify their method to fit whatever HPLC column available in their laboratory instead of looking for the column type best suited to their experiments. Other than that, HPLC column types are often very limited discussed in the class. This is particularly disadvantageous for those who have sufficient access to select the HPLC column they need for the best result. The lack of insight into the types of HPLC columns available in the market also influenced their decision to select the right column in their analysis to a large extent. This article briefly reviews the differences between the commonly used Particle-Packed Columns with the newer yet less frequently used Monolithic Columns. The types of HPLC columns based on polarity, molecular size, and the electrical charge will be described further, along with the working principle of the columns in brief and various examples of options available on the market. Lastly, the uses of HPLC columns in the food industries are also described. This review is expected to contribute to the field of chemical analysis, such as food analysis.

Enantioseparation of basic drugs by reverse phase high-performance liquid chromatography system using carboxymethyl-β-cyclodextrin as chiral mobile phase additive.

A rapid and efficient method was developed for enantioseparation of basic drugs, using carboxymethyl-β-cyclodextrin (CM-β-CD) as chiral mobile phase additive, rather than involving costly chiral column in high-performance liquid chromatography (HPLC) system. Four of the six basic drug enantiomers investigated were successfully separated. The highest resolution reaches 2.15 for threo-(1S,2S)-2-amino-l-p-nitrophenyl-1,3-propanediol. The effects of the organic modifier, pH value, concentration of chiral additive, column temperature, and flow rate of mobile phase on the enantioseparation of analytes were researched. The apparent formation constants of inclusion and the thermodynamic parameters were evaluated to explain the mechanism of chiral recognition.