Sample Solvent as Analyte in High Performance Liquid Chromatography

Abstract

Solvents vary in their behavior in high performance liquid chromatography (HPLC). Water and methanol, among others, are widely used in the mobile phase as well as solvents for the solute. Few reports indicate that the solvent used for the solute can behave as an analyte. Normally, it is generally accepted that the solute solvent, a non-constituent of the mobile phase will be the first eluent. However, a solvent which is a component of the sample can show up as an unexpected peak with its own identity. This solvent may show a similar retention time as some of the unknown components of the sample. This indicates that in some cases the quantitative results may be the sum of the absorptivity of the solute and solvent used for the sample. It is assumed that some solvents show no absorption in the ultraviolet region at which the analysis is being conducted. Depending on the mobile phase composition some solvents can be detected at the wavelengts or wavelengths used for analysis. Water, ethylacetate, and methanol showed absorption at 210 nm when present in the sample being analyzed with a mobile phase of acetonitrile-methanol using a C₁₈ column. These solvents overlapped or showed retention times the same as estriol and testosterone.     

Ethylene Glycol as New Mobile Phase for Resolution of Two-Component Mixture of Cationic Surfactants on Alumina Surface

Aqueous ethylene glycol (ethane 1,2 diol) as a green mobile phase has been used for thin layer chromatographic (TLC) studies of cationic surfactants on alumina layers. Nineteen solvent systems were used to examine the mobility of the surfactants and to discover the best TLC system for the selective separation of dodecyl trimethylammonium bromide (DTAB) from multi-component mixture of other surfactants. Among the TLC systems studied, M₃ (ethylene glycol: water, 8:2) was best for achieving the selective separation of DTAB from multi-component mixture of other surfactants because in this mobile phase mobility of all surfactants except DTAB were insignificant. Effect of organic additives in aqueous ethylene glycol mobile phase on the mobility of surfactants was examined. The results obtained on laboratory made alumina TLC plates and commercially available precoated alumina HPTLC plates were compared. The lower limits of detection of DTAB, CPC, CTAB, HDTAC, and TTAB were 0.02, 0.05, 0.04, 0.06, or 0.08 µg per zone respectively. The resolution of mixture of cationic surfactants was also examined in the presence metal cations as an impurity in the analyzed sample.     

Simultaneous determination of epimers (+)-catechin and (‒)-epicatechin in Onosma bracteatum Wall. using high-performance thin-layer chromatography‒mass spectrometry method

High-performance thin-layer chromatography‒mass spectrometry (HPTLC‒MS) method was developed for the estimation of epimers (+)-catechin (CA) and (‒)-epicatechin (ECA) in Onosma bracteatum Wall. Resolving these epimers is challenging and so method optimization was done for the selection of the stationary phase and the mobile phase to achieve their coherent separation. To further increase the reliability of the obtained densitometric results, HPTLC–MS analysis was performed. The genus Onosma L. is a species-rich genus that exhibits complex patterns of morphological and karyological diversity, and highly debatable taxonomic approaches. Thus, many similar species are described based on morphological differences and often quite ambiguous. To facilitate the identification of O. bracteatum, separation was achieved using pre-coated silica gel 60 F₂₅₄ HPTLC plate as the stationary phase and a mixture of diisopropyl ether–ethyl acetate–formic acid (9.0:0.2:0.7, V/V) as the mobile phase for the separation of epimers CA and ECA. Sample preparation, mobile phase selection and optimization were given importance to manage good resolution (R_F) of these markers. Flavan-3-ols CA and ECA were identified and confirmed on the basis of R_F and in situ UV and MS overlaid spectra with respective standards. The method was validated for linearity, inter-day precision, intra-day precision, repeatability, accuracy, specificity, limit of detection, and limit of quantification. The average recoveries for epimers CA and ECA from ethyl acetate extract fraction (MEF) were found 98.86 and 99.03% indicating the good reproducibility for each marker. The proposed validated HPTLC method is simple, accurate and reproducible and is the first report on the separation and quantification of the epimers CA and ECA in O. bracteatum using HPTLC–MS.

     

Optimization of multicomponent solvent selection in high-performance liquid chromatography using a statistical method

A computer-assisted method is presented for optimization of multicomponent solvent mobile phase selection for separation of O-ethyl-N-isopropyl phosphoro (thioureido) thioates in reversed-phase HPLC and four geometric isomers of pesticides Decis in normal-phase HPLC. The method is based on Snyder's solvent selection triangle concept using a statistical method. The optimization of the separation over the experimental region is based on a special polynomial estimation from seven experimental runs, and resolution (R_s) is used as the selection criterion. Excellent agreement was obtained between predicted data and experimental results.     

Validated HPLC and HPTLC Method for Simultaneous Quantitation of Amlodipine Besylate and Olmesartan Medoxomil in Bulk Drug and Formulation

Two methods are described for simultaneous determination of amlodipine besylate and olmesartan medoxomil in formulation. The first method was based on the HPTLC separation of two drugs on Merck HPTLC aluminium sheets of silica gel 60 F₂₅₄ using n-butanol: acetic acid: water (5:1:0.1, v/v/v) as the mobile phase. The second method was based on the HPLC separation of the two drugs on the RP-PerfectSil-100 ODS-3–C₁₈ column from MZ-Analysetechnik GmbH, Germany and acetonitrile/0.03 M ammonium acetate buffer (pH = 3) in a ratio of 55:45 as the mobile phase. Both methods have been applied to formulation without interference of excipients of formulation.     

A New Quaternary Mobile Phase System for Optimization of TLC Separations of Alkaloids Using Mixture Designs and Response Surface Modelling

Abstract

A new combination of four organic solvents is proposed for the optimization of TLC separations of basic drugs and alkaloids. The solvents are diethylamine (DEA), methanol (MeOH), chloroform (CHCl₃) and ethylacetate (EtAc). They were selected from a collection of ten solvents used in Normal Phase TLC mobile phases recommended for the separation of alkaloids and basic drugs in the literature. The selection was based on the classification of solvents according to selectivity and solubility parameters. Excluded were apolar and weak solvents that show no selective (polar) properties and are used only for the adjustment of the solvent strength. Polar solvents from different selectivity groups were selected to combine as many as possible selective effects in one solvent system. The final choice was made considering the displacement theory for Liquid Solid Chromatography.     

Simultaneous Determination of Tinidazole and Furazolidone in Suspension by HPTLC and HPLC

Abstract

High performance thin layer chromatographic (HPTLC) and high performance liquid chromatographic (HPLC) methods were developed for the simultaneous determination of Tinidazole and Furazolidone in suspension.

In the HPTLC method the separation of Tinidazole and Furazolidone was carried out on silica gel 60F₂₅₄ HPTLC glass plate using chloroform:methanol:ammonia (9:1:0.1 v/v) as a mobile phase. Rf values obtained were 0.63 and 0.79 for Furazolidone and Tinidazole respectively. Densitometric evaluation was done at 335 nm. Linearity was obtained within the concentration range 10–50 μg/ml and 3.5–17.5 μg/ml for Tinidazole and Furazolidone respectively.

The second method is based on high performance liquid chromatography on a reversed phase column (μ Bondapak C₁₈) using a mobile phase comprised of water: acetonitrile: triethylamine (80:20:0.1 v/v) adjusted to pH = 3.0 with dil. phosphoric acid. Retention times were 5.24 and 7.82 min for Tinidazole and Furazolidone respectively at a flow rate of 1.5 ml/min. Detection was done at 335 nm. Linearity was obtained within the concentration range 30–180 μg/ml and 10.5–63 μg/ml for Tinidazole and Furazolidone resp.     

Advanced simplex optimization of two-factor selectivity by high performance thin-layer chromatography

Summary A computer-assisted mixture design simplex method is presented for optimization of the separation of a mixture of five benzoic acid derivatives in reversephase HPTLC and twelve PTH-amino acids in normal phase HPTLC. A two-factor selectivity rectangle concept was used. The method is based on a special polynomial estimated from nine experimental runs, using the R_F difference as the selection criterion, with connection to a general simplex method for optimization using a microcomputer. Excellent agreement is obtained between predicted data and experimental results, and more than half the number of experiments required in the general simplex method can be omitted.     

“PRISMA” Model for Computer-Aided HPLC Mobile Phase Optimization Based on an Automatic Peak Identification Approach

Abstract

The tripartite “PRISMA” optimization model, as part of the “PRISMA” system, includes all possible solvent combinations between 1–4 solvents, with a possible fifth one as modifier. The solvent composition is characterized by the solvent strength (S_T) and the selectivity points (P_S).

At a constant S_T the correlation between the P_S and the retention data (horizontal function) can be described by a quadratic function. For constant P_S the solvent strengths and retention data correlate (vertical function) with a logarithmic function. These correlations are used to formulate a mathematical model for the dependence of retention times (capacity factor) on the mobile phase composition. Unknown compounds are estimated in the mathematical model from a sequence of standard chromatograms after having identified individual peaks by an automatic procedure. Only retention times, relative peak areas, and information about the mobile phase compositions are required as input for the identification approach. The approach involves a combination of statistical methods which exploit both the basic properties of retention data and the mathematical relation between retention data, selectivity points, and solvent strength as derived from the “PRISMA” model. Diagnostic information for checking the identification is generated as a by-product. The mathematical model completed by the estimated constants predicts the expected retention times for each possible mobile phase combination. Peak start and peak end times are predicted in a way similar to the retention times, once the identification has been performed. The most important aspects of a chromatogram can thus be predicted for arbitrary mobile phases.

The separation quality of predicted chromatograms is assessed by the chromatographic response function (CRF). The optimal mobile phase combination is that which theoretically generates the chromatogram with the maximal CRF value. This optimal composition is found by a simple mathematical procedure, which maximises the CRF in dependence upon the mobile phase combination. The optimum found is a local one if the starting set of chromatograms contains no variation of the solvent strength, and a global one if, in the set of starting chromatograms, the solvent strength is varied in a suitable way. Recommendations for the starting position are given.

Twelve measurements are necessary for a local optimum, and 15 for the global one. To increase the accuracy, six measurements at three different solvent strength levels are proposed. Generally the highest and the lowest solvent strength level differ by ±(5)% from the middle level.

This strategy is also relevant when modifiers are used in constant amounts. The chromatographic behavior of substances to be separated can be predicted with 1% accuracy from correlations of k' values and selectivity points. Based on these relationships, an automatical mobile phase optimization strategy for isocratic separations is suggested with the “PRISMA” model.     

Simultaneous Separation and Determination of Lamivudine and Zidovudine in Pharmaceutical Formulations Using the HPTLC Method

Abstract

A high-performance thin-layer chromatographic method (HPTLC) for the simultaneous determination of lamivudine and zidovudine in a binary mixture has been developed. The method developed was based on HPTLC separation of the two drugs followed by densitometric measurements of spots at 276 and 271 nm for lamivudine and zidovudine, respectively. Separation was carried out on Merck HPTLC silica-gel 60 F₂₅₄ plates, using toluene/chloroform/methanol (1:6:3 v:v) as the mobile phase. Validation of the method was performed based on The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines in terms of linearity, accuracy, precision, limit of detection, limit of quantification, and robustness. Second-order polynomial equations were obtained for the regression line in the ranges of 250–1400 and 250–1700 ng/spot for lamivudine and zidovudine respectively. Correlation coefficient (r) values were 0.9998 for both analytes. The method provides sufficient accuracy as indicated by recovery percentages given for lamivudine and zidovudine. For system precision study, the low coefficient of variation values (<2%) for both lamivudine and zidovudine ensured reproducible performance of the instrument. In the method precision study, coefficients of variation <2% were obtained, which showed that the proposed method provides acceptable intraday and interday variation. The detection and quantification limits and were 3.06 and 9.28 ng/spot for lamivudine and 3.34 and 10.13 ng/spot for zidovudine, respectively. Parameters such as mobile-phase composition, volume of mobile phase, time from spotting to development, and time from development to scanning were employed while testing for robustness of the method, and the standard deviation of peak areas was calculated for each parameter. The low coefficient of variation values indicated the robustness of the method. Statistical manipulation did not show any significant effect of one parameter over the others on the robustness of the method.     

Separations in Thin Layer and High Performance Liquid Chromatography Using Alkyl Silica Gel Bonded Phases

Abstract

Separations in thin layer (TLC) and high performance liquid chromatography (HPLC) using alkyl bonded phases were carried out under optimum solvent conditions for each of three phases, RP-2, RP-8 and RP-18. The phases were tested for their efficiency and resolving power using three groups of compounds in three binary organic-water mobile phases. The organic solvents were acetonitrile, methanol and tetrahydrofuran, which are widely used as solvent modifiers in reversed phase liquid chromatography.

The results showed that, unlike HPLC, TLC using RP-18 plates was the most, and RP-2 plates the least, efficient. A naphthalene and biphenyl mixture which was resolved by HPLC using any of the three solvents and columns was not resolved by TLC using any plate or solvent combination, unless the plate was prewashed with an organic modifier. The addition of NaCl (1–2% wt/vol) to the solvent for TLC speeded development unless an alcohol was used, but did not greatly affect the separation.     

Effect of Alkyl Chain Length of Bonded Silica Phases on Separation,Resolution and Efficiency in High Performance Liquid Chromatography

Abstract

A comparative study of alkyl bonded phases was carried out under optimum solvent conditions for each phase. Three columns, RP-2, RP-8 and RP-18, were tested for their efficiency and resolving power using three groups of compounds in three binary organic-water mobile phases. The organic solvents were acetonitrile, methanol and tetrahydrofuran which are widely used as solvent modifiers.

The results indicate that each of the three factors, i.e. solvent, solute and bonded alkyl chain length, play an important role, with the solvent being the most significant. When tetrahydrofuran-water was used as the mobile phase, the ratio of THF/H₂O did not vary by much when an RP-2, RP-8 or RP-18 column was used to separate naphthalene from biphenyl, dimethylphthalate from diethylphthalate or anthraquinone from methyl, anthraquinone and ethyl anthraquinone. When acetonitrile-water and methanol-water were used the ratio of organic modefier to water changed so as to accomodate the hydrophobic properties of the columns. The efficiency of the columns, expressed as theoretical plates per meter (TPM) was highest when acetonitrile-water was used as the mobile phase. Although there were variations in TPM and resolution from column to column, the three columns gave good separation of the components of the three groups of compounds.     

Ion Interaction Chromatographic Separation of Amino Acids Using a Basic-Tetraalkylammonium Salt Mobile Phase

Abstract

A basic mobile phase containing a tetraalkylammonium (R₄N⁺) salt was used to enhance the retention of free amino acids (AA) in their anion form on a polystyrene divinylbenzene copolymeric (Hamilton PRP-1) nonpolar stationary phase adsorbent. Major variables, which can be readily manipulated to alter this retention and resolve complex AA mixtures, are: structure and concentration of R₄N⁺ salt, type and amount of organic modifier in the mobile phase solvent, concentration and selectivity of the counteranion present, and mobile phase pH and ionic strength. Mobile phase gradients based on a pH change, or an ionic strength change and their combination, while all other variables are constant, were evaluated for the separation of complex AA mixtures. Detection was accomplished by absorbance or fluorescence after a post-column ortho-phthalaldehyde reaction.     

Instrumental HPTLC of Aflatoxins: Fluorescence Enhancement by Corn Free Fatty Acids

Abstract

Greater than 100% recoveries using instrumental HPTLC were observed for aflatoxin (AFT) analysis in spiked corn samples. I₂ detection of spots over-laying AFT B₁ and B₂ were identified by GLC as C₁₆ ^?c18 free fatty acids (FFA). These FFA were found to enchance the fluorescence of AFT B₁ from 13.7% to 35.7% greater than controls resulting in >100% recoveries. The inclusion of glacial acetic acid in the TLC mobile phase resulted in an increased mobility of the FFA which eliminated the positive interference on AFT fluorescence. Recoveries using the modified developing solvent then gave values in acceptable ranges.     

Critical assessment of three high performance liquid chromatography analytical methods for food carotenoid quantification

Three sets of extraction/saponification/HPLC conditions for food carotenoid quantification were technically and economically compared. Samples were analysed for carotenoids α-carotene, β-carotene, β-cryptoxanthin, lutein, lycopene, and zeaxanthin. All methods demonstrated good performance in the analysis of a composite food standard reference material for the analytes they are applicable to. Methods using two serial connected C₁₈ columns and a mobile phase based on acetonitrile, achieved a better carotenoid separation than the method using a mobile phase based on methanol and one C₁₈-column. Carotenoids from leafy green vegetable matrices appeared to be better extracted with a mixture of methanol and tetrahydrofuran than with tetrahydrofuran alone. Costs of carotenoid determination in foods were lower for the method with mobile phase based on methanol. However for some food matrices and in the case of E–Z isomer separations, this was not technically satisfactory. Food extraction with methanol and tetrahydrofuran with direct evaporation of these solvents, and saponification (when needed) using pyrogallol as antioxidant, combined with a HPLC system using a slight gradient mobile phase based on acetonitrile and a stationary phase composed by two serial connected C₁₈ columns was the most technically and economically favourable method.     

The Effect of Binary Solvent Composition and Polarity on Separations in Reversed Phase Thin Layer and High Performance Liquid Chromatography

Abstract

The effect of the solvent's composition and polarity on separation in reversed phase thin layer and high performance liquid chromatography is discussed. These results show that retention times cannot be predicted merely from the polarity of the binary mobile phase. Although organic modifiers with the same physico-chemical properties and from the same solvent group were used, the retention times obtained using binary mobile phases having the same polarity, were different. It was also observed that normal chain carbon alcohols gave retention times different from those with a branched chain (n-propanol vs. iso-propanol), and the longer the alcohol chain the higher the R_f value. The results also show that not only the organic modifier used is important but the solute mixture used.     

Utilization of a Systematic Solvent Selection Method for the HPLC Determination of a Trace Isoheric Contaminant

Abstract

A systematic solvent selection method is used for the development of HPLC conditions for the separation and detection of trace amounts of threo isomer in samples of the bronchodilating drug procaterol, (±)-(R?, S?)-8-hydroxy-5-[1-hydroxy-2-[(1-utethyl-e thyl)amino]butyl]-2(1H)-quinoline (erythro isomer). The method involves the chromatography of a mixture of the two isomers using seven different mixtures of three base solvents as mobile phases and the mapping of resolutions using a computer-generated mathematical model. The optimized mobile phase predicted by the model gives excellent resolution between the two isomers at levels as low as 1% of the threo compound.     

Quick and Simple Determination of Dihydroergotamine by High-Performance Liquid Chromatography

Abstract

A simple and rapid liquid chromatographic assay method using a fluorescence detector for quantitation of dihydroergotamine in plasma without extraction was developed. After precipitating the protein with acetonitrile, the supernatant liquid was directly injected for analysis. Chromatographic separation was achieved on C₁₈ reversed phase column and the mobile phase was the isocratic mixture of methanol, acetonitrile and glycine buffer (0.5:3.5:6.0). With this eluting solvent the drug and its internal standard were well separated from the interference of the plasma sample. The average recovery of dihydroergotamine from 6 replicate samples of different concentrations (5-30 ng/ml) were 92.2 ± 3.37%. The minimum amount of dihydroergotamine detectable by this method was 2 ng/ml of sample.     

HPLC separation of isomers of tetrahydro-1,5-benzothiazepines and tetrahydro-1,5-benzodiazepines

Summary Separation of positional and cis-trans-isomers of tetrahydro-1,5-benzothiazepines and tetrahydro-1,5-benzodiazepines was studied using reversed-phase chromatography and liquid-solid chromatography. The selection of solvent was based on the selective triangle for solvents. Systems of separation consisted of C₁₈ columns and methanol, THF or acetonitrile in water for the reversedphase method; it was suitable for the separation of positional isomers only but the liquid-solid method was suitable for separation of cis-trans-isomers as well as positional isomers using a silica column and ethyl ether, chloroform or ethyl acetate as the mobile phase respectively.