Improving the universal response of evaporative light scattering detection by mobile phase compensation

Mobile phase compensation, first reported for the charged aerosol detector (CAD), was used as a suitable method to overcome problems related to the mobile phase-dependent response of the evaporative light scattering detector (ELSD). Mobile phase compensation was effectively performed both in the flow injection- and in gradient modes. Without compensation, the response factors of the ELSD for six sulfonamide drugs differed by a factor of two when varying the mobile phase composition between 10 and 90% acetonitrile. This change could be effectively eliminated using the technique of mobile phase compensation, where a secondary pump with a reversed gradient was used to provide the detector with a constant composition of the mobile phase. For identical experimental conditions, the ELSD showed a nearly constant, albeit somewhat reduced, response with compensation. This indicates that under such conditions, the ELSD behaved as a concentration-sensitive detector. The analysis of sulfonamides drugs at 0.05% level using gradient UPLC-ELSD separation with mobile phase compensation is demonstrated.     

Determination of UV-transparent compounds by liquid chromatography using the UV detector

An indirect UV photometric detection technique is described in which a low concentration of a UV-absorbing compound (UVAC) is added to the mobile phase in reversed phase liquid chromatography, thereby making it possible for non UV-absorbing compounds such as the lower alcohols to be detected by the UV detector. This happens because the injected analyte may extract a portion of the UV absorbing compound from the mobile and/or stationary phase and the complex is co-eluted as a positive peak at the retention time of the analyte. Alternatively, the injected analyte may appear as a negative peak if the UV-absorbing compound is transferred to the mobile and stationary phases. In any case, the injected compound appears either as a positive or negative peak depending on the relative polarities and concentrations of all the compounds in the system. In addition, the resulting excess or deficiency of detection agent in the stationary phase is eluted separately as a positive or negative peak, indicating that the system has returned to equilibrium. In the work described herein, the chromatographic conditions and variables of the indirect photometric technique were studied to develop a quantitative HPLC method for UV-transparent compounds. It was found that under optimal conditions it is possible to determine some analytes quantitatively at concentrations as low as 0.05%.     

Indirect conductimetric detection of amino acids after liquid chromatographic separation

A liquid chromatographic method using indirect conductimetric detection is proposed for the determination of low levels of organic compounds, which does not require any special functional characteristics of the analyte. The signal detected is proportional to the molar concentration of the analyte and independent of its nature. The detector response is linearly dependent on analyte concentrations over at least three orders of magnitude. The basis of the detection is to create a conducting background, which will decrease on elution of the organic compounds. The theory of the method is discussed, with special reference to the quantitative displacement of the conducting species of the mobile phase from the column by the analyte on sample injection. The proposed method has been applied to study the chromatographic behaviour of twenty-one amino acids, where a 5 -μm Econosil CN column was used as the stationary phase with a mixture of water-acetonitrile-tetrahydrofuran (70:20:3) containing 1 mM perchloric acid or trichloroacetic acid as the mobile phase. The proposed method allows as little as 10 ng of each amino acid to be determined.     

A comparative study of commercial liquid chromatographic detectors for the analysis of underivatized amino acids

This study compares the main commercial detectors that can detect amino acids in their underivatized form. The detectors tested are: the chemiluminescent nitrogen detector (CLND), the evaporative light scattering detector (ELSD), the nuclear magnetic resonance spectrometer, conductivity detector, refractive index, UV, and electrospray quadrupole mass spectrometry (in simple and tandem MS mode). As ELSD, CLND and MS require a volatile mobile phase, an ion-pair reversed-phase liquid chromatographic system was selected, consisting of an octadecyl column and an aqueous mobile phase containing pentadecafluorooctanoic acid as volatile ion-pairing reagent. Underivatized taurine, hypotaurine, aspartic acid, hydroxyproline, asparagine, serine, glycine, glutamine, cysteine, glutamic acid, threonine and alanine were simultaneously analysed with each detector. In order to test the applicability of these detectors to "real world" samples, the amino acid stoichiometry of the tetrapeptide Gly-Gly-Asp-Ala was determined with each detector after acid hydrolysis. The detectors were compared in terms of linearity, limit of detection, advantages and disadvantages as well as special features (capacity to provide structural information, specificity, quantification with single calibration curve, etc.).     

Determination of Biodiesel and Triacylglycerols in Diesel Fuel by LC

A high performance liquid chromatographic method was developed for quantifying blends of biodiesel (simple alkyl esters of fatty acids) in petrodiesel. The method uses a silica column with an isocratic mobile phase consisting of hexane and methyl t-butyl ether. Separated components were quantitated using either an evaporative light scattering detector (ELSD) or UV detector. Precision of injection and linearity of response of the ELSD and UV detectors over a range of biodiesel-petrodiesel blends [1–30 v/v %] were established by use of standards. The method also can be used for quantitating similar levels of oils or fats (triacylglycerols) in petrodiesel.     

Mechanism of response enhancement in evaporative light scattering detection with the addition of triethylamine and formic acid

Triethylamine with an equimolar amount of formic acid added to the mobile phase provides an enhancement of the evaporative light scattering detector (ELSD) response. After characterization of the influence of various parameters on the ELSD response, a sequential strategy was defined to elucidate this response enhancement. The response enhancement was more marked at low mobile phase flow rate, and was highly dependent on solutes and solvents. The influence of drift tube temperature on response enhancement with various solutes demonstrated that triethylamine and formic acid mainly act as mass amplifiers by the inclusion of triethylamine-formic acid clusters inside the droplets.     

Reductive Mode Thin-Layer Amperometric Detector for Liquid Chromatography

Abstract

An electrochemical detector for liquid chromatography is described based on the use of a mercury film electrode in a thin-layer cell with the auxiliary electrode placed across the channel from the working electrode. Considerations relevant to optimizing performance of the detector are described including removal of dissolved oxygen from the sample and mobile phase. Detection limits are reported for a number of reducible organic compounds of interest in environmental and biomedical research. Detection limits at the picomole level are readily achieved for easily reduced nitro compounds. Typical applications are illustrated for the determination of parathion in field runoff water and chloramphenicol in human plasma.     

Universal response model for a corona charged aerosol detector

The universality of the response of the Corona Charged Aerosol Detector (CoronaCAD) has been investigated under flow-injection and gradient HPLC elution conditions. A three-dimensional model was developed which relates the CoronaCAD response to analyte concentration and the mobile phase composition used. The model was developed using the response of four probe analytes which displayed non-volatile behavior in the CoronaCAD and were soluble over a broad range of mobile phase compositions. The analyte concentrations ranged from 1μg/mL to 1mg/mL, and injection volumes corresponded to on-column amounts of 25ng to 25μg. Mobile phases used in the model were composed of 0-80% acetonitrile, mixed with complementary proportions of aqueous formic acid (0.1%, pH 2.6). An analyte set of 23 compounds possessing a wide range of physicochemical properties was selected for the purpose of evaluating the model. The predicted response was compared to the actual analyte response displayed by the detector and the efficacy of the model under flow-injection and gradient HPLC elution conditions was determined. The average error of the four analytes used to develop the model was 9.2% (n=176), while the errors under flow-injection and gradient HPLC elution conditions for the evaluation set of analytes were found to be 12.5% and 12.8%, respectively. Some analytes were excluded from the evaluation set due to considerations of volatility (boiling point <400°C), charge and excessive retention on the column leading to elution outside the eluent range covered by the model. The two-part response model can be used to describe the relationship between response and analyte concentration and also to offer a correction for the non-linear detector response obtained with gradient HPLC for analytes which conform to the model, to provide insight into the factors affecting the CoronaCAD response for different analytes, and also as a means for accurately determining the concentration of unknown compounds when individual standards are not available for calibration.     

The advantages of evaporative light scattering detection in pharmaceutical analysis by high performance liquid chromatography and supercritical fluid chromatography

The evaporative light scattering detector (ELSD) has been used in pharmaceutical analysis by liquid and supercritical fluid chromatography. An ELSD equipped with interchangeable interfaces enables the use of various eluents (UV- or non-UV-absorbing) in isocratic or gradient mode. Analyses were performed on several non-UV-absorbing excipients and active substances. The narrow spread of the response factors of the various compounds investigated has indicated that the detector is suitable for direct raw quantitation of unknown samples in stability studies.     

Comparison of two aerosol-based detectors for the analysis of gabapentin in pharmaceutical formulations by hydrophilic interaction chromatography

Comparison of hydrophilic interaction chromatography (HILIC) columns coupled with an evaporative light scattering detector (ELSD) or charged aerosol detector (CAD) was done for the detection of gabapentin in pharmaceutical formulations. The chromatographic separations were achieved on four HILIC columns: ZIC HILIC, ZIC pHILIC, Luna HILIC, and Atlantis HILIC. Experimental factors such as mobile phase composition, acetonitrile content, and mobile phase pH were evaluated. Validation of method was done in terms of linearity, sensitivity, accuracy, and precision. The performance of ELSD detection method is comparable to that of CAD. The intra-day and inter-day variations were below 1.7% and 3.2% for CAD and 2.8%, and 3.4% for ELSD, respectively. In addition, detection sensitivities of ELSD, CAD, and UV detectors were also compared for HILIC and reversed phase (RP) modes and the highest sensitivities were obtained in the HILIC mode when connected with CAD and ELSD. The developed HILIC aerosol based detection methods were successfully applied to the analysis of gabapentin in commercial tablets and capsules.     

高效液相色谱-蒸发光散射检测法直接检测20种未衍生基本氨基酸

应用国产蒸发光散射检测器(ELSD),建立了一种采用反相高效液相色谱-蒸发光散射检测器(RHPLC-ELSD)直接测定20种未衍生基本氨基酸的分析方法,并将其用于氨基酸注射液中氨基酸含量的测定。采用BISCHOFFTM　C18 AQ PLUS色谱柱(250 mm×4.6 mm, 5 μm)分离,以甲醇-0.2%七氟丁酸溶液(含0.1%三氟乙酸)为流动相进行梯度洗脱,流速0.8 mL/min,ELSD飘移管温度40 ℃,载气流量2.5 L/min,对20种基本氨基酸进行分离检测。氨基酸的质量浓度在30～300 mg/L范围内,其峰面积的对数值与进样质量的对数值呈良好的线性关系;氨基酸的检出限(信噪比(S/N)＞3)介于24 ～100 ng之间,样品加标回收率为90.6%～106.0%。结果表明,该系统及方法操作简便快速、准确可靠,无需依靠专门的氨基酸分析仪或衍生处理氨基酸即可直接测定氨基酸注射液中氨基酸含量,为药品、食品及化工生产等领域混合氨基酸样品的直接检测提供了参考。     

Analysis of triacylglycerols on porous graphitic carbon by high temperature liquid chromatography

The retention behaviour of several triacylglycerols (TAGs) and fats on Hypercarb, a porous graphitic carbon column (PGC), was investigated in liquid chromatography (LC) under isocratic elution mode with an evaporative light scattering detector (ELSD). Mixtures of chloroform/isopropanol were selected as mobile phase for a suitable retention time to study the influence of temperature. The retention was different between PGC and non-aqueous reversed phase liquid chromatography (NARP-LC) on octadecyl phase. The retention of TAGs was investigated in the interval 30-70 degrees C. Retention was greatly affected by temperature: it decreases as the column temperature increases. Selectivity of TAGs was also slightly influenced by the temperature. Moreover, this chromatographic method is compatible with a mass spectrometer (MS) detector by using atmospheric pressure chemical ionisation (APCI): same fingerprints of cocoa butter and shea butter were obtained with LC-ELSD and LC-APCI-MS. These preliminary results showed that the PGC column could be suitable to separate quickly triacylglycerols in high temperature conditions coupled with ELSD or MS detector.     

Direct analysis of plasma samples for drug discovery compounds using mixed-function column liquid chromatography tandem mass spectrometry

A sensitive, efficient, high throughput, direct injection bioanalytical method based on a single column and high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) was developed for pharmacokinetic analysis of early drug discovery compounds in plasma samples. After mixing with a working solution containing an internal standard each plasma sample was directly injected into a polymer-coated mixed-function column for sample cleanup, enrichment and chromatographic separation. The stationary phase incorporates hydrophilic polyoxyethylene groups and hydrophobic groups to the polymer-coated silica. This allows proteins and macromolecules to pass through the column due to restricted access to the surface of the packing while retaining the drug molecules on the bonded hydrophobic phase. The analytes retained in the column with a largely aqueous liquid mobile phase were then chemically separated by switching to a strong organic mobile phase. The column effluent was diverted from waste to the mass spectrometer for analyte detection. Within 200 plasma sample injections the response ratio (analyte vs. internal standard, %CV = 4.6) and the retention times for analyte and internal standard were found consistent and no column deterioration was observed. The recoveries of test compound in various plasma samples were greater than 90%. The total analysis time was 相似文献   

Comparison of the response of four aerosol detectors used with ultra high pressure liquid chromatography

The responses of four different types of aerosol detectors have been evaluated and compared to establish their potential use as a universal detector in conjunction with ultra high pressure liquid chromatography (UHPLC). Two charged-aerosol detectors, namely Corona CAD and Corona Ultra, and also two different types of light-scattering detectors (an evaporative light scattering detector, and a nano-quantity analyte detector [NQAD]) were evaluated. The responses of these detectors were systematically investigated under changing experimental and instrumental parameters, such as the mobile phase flow-rate, analyte concentration, mobile phase composition, nebulizer temperature, evaporator temperature, evaporator gas flow-rate and instrumental signal filtering after detection. It was found that these parameters exerted non-linear effects on the responses of the aerosol detectors and must therefore be considered when designing analytical separation conditions, particularly when gradient elution is performed. Identical reversed-phase gradient separations were compared on all four aerosol detectors and further compared with UV detection at 200 nm. The aerosol detectors were able to detect all 11 analytes in a test set comprising species having a variety of physicochemical properties, whilst UV detection was applicable only to those analytes containing chromophores. The reproducibility of the detector response for 11 analytes over 10 consecutive separations was found to be approximately 5% for the charged-aerosol detectors and approximately 11% for the light-scattering detectors. The tested analytes included semi-volatile species which exhibited a more variable response on the aerosol detectors. Peak efficiencies were generally better on the aerosol detectors in comparison to UV detection and particularly so for the light-scattering detectors which exhibited efficiencies of around 110,000 plates per metre. Limits of detection were calculated using different mobile phase compositions and the NQAD detector was found to be the most sensitive (LOD of 10 ng/mL), followed by the Corona CAD (76 ng/mL), then UV detection at 200 nm (178 ng/mL) using an injection volume of 25 μL.     

Exploration of the effects of chloride ions on the analysis of polar compounds at low concentrations by hydrophilic interaction liquid chromatography coupled to a charged aerosol detector: Application to tromethamine

In this study, we discuss the origin of the slightly increased response of the charged aerosol detector when low-concentration polar drugs formulated with sodium chloride are analyzed by hydrophilic interaction liquid chromatography coupled to the charged aerosol detector. In the case of tromethamine mixed with saline solutions, we investigated several levels including the mobile phase, sample matrix, and detection. We show that the analysis of the rich-salted sample results in both interactions with the mobile phase modifiers and the stationary phase during the run time. With 150 mM NaCl as a compounding solution, a slight increase in the tromethamine peak area was observed (<5.5%). Our study suggests that chloride ions in excess sequentially interact firstly with the counterions from the organic modifiers and secondly with the analyte via the stationary phase and the contribution of hydrophilic interaction liquid chromatography retention mechanisms. Because of these effects, the hydrophilic interaction liquid chromatography-charged aerosol detector analysis of drugs in saline solutions requires particular attention, and a correction factor for quantitative purposes that accounts for formulation ions remains appropriate.     

Determination of seven compounds in Tang Maikang granule by high-performance liquid chromatography

An accurate and sensitive high-performance liquid chromatography method is developed and applied to the determination of seven compounds in a kind of traditional Chinese medicinal preparation of Tang Maikang Granule. The method is performed on Hypersil C(18) column (250- x 4.6-mm i.d., 5 microm), and different mobile phases and detectors are selected according to the various compounds. For astragaloside IV, an evaporative light scattering detector (ELSD) is used with a gradient of methanol-water at an eluent gas rate of 2.0 mL/min, under a drift tube temperature of 80 degrees C. Formononetin and calycosin are also eluted by a gradient of methanol-water, but a photodiode array (PDA) detector is used at a wavelength of 254 nm for formononetin and calycosin. A PDA detector at a wavelength of 230 nm is used for paeoniflorin, with methanol-water (30:70, v/v) as the mobile phase. For danshensu and protocate chualdehyde, an eluent of methanol-0.5% acetic acid (12:88, v/v) is used, with PDA detection at 280 nm. For berberine, methanol and water containing 0.1% sodium dodecanesulphonate (SDS) and 0.1% phosphorous acid (70:30, v/v) is employed as the mobile phase, also using a PDA detector, but the detection wavelength is 265 nm. The intra- and interrun precision (relative standard deviation) of this method is less than 5% for seven analytes.     

高效液相色谱/挥发激光散射检测器测定奶粉和鸟粪中的甾体化合物

利用反相高效液相色谱／挥发激光散射检测器发展了分析甾体化合物的新方法,采用甲醇和水溶液为冲洗体系,梯度淋洗,12种甾体类化合物在35min内得到很好的分离。方法简单快速,不需衍生,样品经固相萃取预处理,回收率介于84.0％～102.8％之间,响应值和浓度不呈简单的线性关系,在5～50mg／L浓度范围内对峰面积与浓度进行对数线性回归分析,得回归系数大于0.99。     

Factors affecting C:H and C:N ratios determined by gas chromatography coupled with atomic emission detection

An atomic emission detector has been evaluated for the estimation of C:H and C:N ratios of compounds eluted in gas chromatography. When C:H and C:N ratios were estimated using external reference compounds, the greatest accuracy was obtained when the quantity of the reference compound was similar to that of the analyte of interest. The accuracy of the C:H and C:N ratios determined was also affected by the molecular structure of the reference compound. C:H ratios of the greatest accuracy were obtained by using reference compounds having structures similar or close to those of the compound of interest; this was not always so for C:N ratios.     

Investigation of polar organic solvents compatible with Corona Charged Aerosol Detection and their use for the determination of sugars by hydrophilic interaction liquid chromatography

A range of organic solvents (ethanol, isopropanol and acetone) has been investigated as alternatives to acetonitrile and methanol when used in conjunction with Corona Charged Aerosol Detection (Corona CAD). These solvents have been evaluated with regard to their effect on the response of the Corona CAD. Three dimensional response surfaces were constructed using raw data showing the relationship between detector response, analyte concentration and percentage of organic solvent in the mobile phase, using sucralose or quinine as the test analyte. The detector response was non-linear in terms of analyte concentration for all solvents tested. However, detector response varied in an approximately linear manner with percentage of organic solvent over the range 0–40% for ethanol or isopropanol and 0–80% for acetone and methanol. The chromatographic performance of the various solvents when used as aqueous–organic mobile phases was evaluated for isocratic and gradient separations of sugars and sugar alcohols by hydrophilic interaction liquid chromatography (HILIC) using an Asahipak NH2P-504E column coupled with Corona CAD detection. It was found that whilst acetonitrile provided the highest column efficiencies and lowest detection limits of the solvents studied, acetone also performed well and could be used to resolve the same number of analytes as was possible with acetonitrile. Typical efficiencies and detection limits of 5330 plates m⁻¹ and 1.25 μg mL⁻¹, respectively, were achieved when acetone was used as the organic modifier. Acetone was utilised successfully as an organic modifier in the HILIC separation of carbohydrates in a beer sample and also for a partially digested dextran sample.