Analysis of the essential oil from radix bupleuri using capillary gas chromatography

A simple and rapid capillary gas chromatographic (CGC) method with flame ionization detection has been newly developed for analysis of the essential oil from Radix Bupleuri. Twenty components were identified with gas chromatography-mass spectrometry. E-2-heptenal, furan, 2-pentyl, and E-2-nonenal were quantified simultaneously using the internal standard method. Decane was used as an internal standard. Separation and quantification were achieved on a DB-5 capillary column (30 m x 0.25 mm i. d., 0.25-microm film thickness). The oven temperature was programmed as follows: 60 degrees C to 70 degrees C at 1 degree C/min rate, 70 degrees C for 10 min, 3 degrees C/min to 120 degrees C, 20 degrees C/min to 250 degrees C, and held at 250 degrees C for 5 min. The oven pressure was programmed as follows: 46.1 kPa for 25 min, 20.0 kPa/min to 77.6 kPa, and then held for 22 min. Split injection was conducted with a split ratio of 10:1; flow-rate, 1.00 ml/min; carrier gas, nitrogen; injector temperature, 280 degrees C; and detector temperature, 280 degrees C. The system proved effective in resolving E-2-heptenal, furan, 2-pentyl, and E-2-nonenal peaks from their interfering components. The method displayed excellent linearity in the range of 26.8-1072 microg/ml (E-2-heptenal), 6.5-1292 microg/ml (furan, 2-pentyl), and 7.8-1564 microg/ml (E-2-nonenal). The average recovery rates of E-2-heptenal, furan, 2-pentyl, and E-2-nonenal were 100.3%, 102.8%, and 97%, respectively. CGC is a quick and accurate method for analysis of the essential oil from Radix Bupleuri.     

Analysis of neolignans compounds of Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck by HPLC

A high performance liquid chromatographic (HPLC) method was developed and validated for quantitative determination of neolignans in extracts of Piper regnellii var. pallescens. The analysis were carried out on a Metasil ODS column (150 mm x 4.6 mm, 5 microm) at 30 degrees C, using as mobile phase acetonitrile-water (60:40, v/v) containing 2% acetic acid. The flow rate was 1.0 ml/min and the detection was at 280 nm. The validation using conocarpan as standard demonstrated that the method presents linearity (linear correlation coefficient=0.9991), precision (relative standard deviation <5%) and accuracy (mean recovery=104.55%) in the concentration range 31.25-500 microg/ml. The limit of detection (LOD) was 1.68 microg/ml and the limit of quantitation was 5.60 microg/ml. This method allowed the identification and quantification of conocarpan, eupomatenoid-5 and eupomatenoid-6 in the hydroethanolic extracts obtained from the leaves, stems and roots by maceration process. All the extracts showed the same chromatographic profile, being that the extract of the roots presented the highest concentration of neolignans.     

Development and validation of a liquid chromatographic method for determination of enantiomeric purity of citalopram in bulk drugs and pharmaceuticals

A simple, rapid and robust LC method for enantiospecific separation and determination of citalopram in drugs and pharmaceuticals was developed using UV and polarimetric detectors connected in series. Baseline separation with resolution > or = 3.0 was achieved within 20 min on Chiralcel OD-H (250 mm x 4.6 mm) 5 microm column using a mobile phase containing of n-hexane:2-propanol:triethylamine (TEA) (95:05:0.1 v/v/v) at a flow rate of 1.0 ml/min at 25 degrees C. Effects of 2-propanol, triethylamine and temperature on enantioselectivity and resolution of the enantiomers were evaluated. Clopidogrel hydrogen sulphate was used as an internal standard (IS) for quantitative determinations using UV detector at 240 nm. Polarimetric detector was used for identification of enantiomers. The limits of detection (LOD) and quantification (LOQ) were 0.5 and 1.3 microg/ml respectively for both the enantiomers. The linearity of the method was in the range of 50-600 microg/ml with r2 > 0.9999. The inter- and intra-day assay precision was less than 0.63% (%R.S.D.) and recoveries were in the range 99.38-100.41%. The method was validated and found to be suitable for determination enantiomeric purity of citalopram in bulk drugs and pharmaceutical formulations.     

First-derivative spectrophotometric and LC determination of cefuroxime and cefadroxil in urine

Two methods are presented for the determination of cefuroxime and cefadroxil in human urine using first (1D) derivative spectrophotometry and high-performance liquid chromatography. Cefuroxime and cefadroxil were determined by measurement of their first-derivative amplitude in 0.1 N sodium hydroxide at 292.5 and 267.3 nm, respectively in the concentration range of 2-10 microg ml(-1) for each drug. The HPLC method depends upon using a LiChrospher 100 RP-18 (5 microm) column at ambient temperature for cefuroxime and 35 degrees C for cefadroxil with mobile phases consisting of water-acetonitrile-acetic acid (85:15:0.1 v/v) at a flow rate of 1.5 ml min(-1) for cefuroxime; and 0.02 M potassium dihydrogen phosphate-acetonitrile (95:5 v/v) containing 0.003% (w/v) hexanesulphonic acid sodium salt and adjusted to apparent pH 3 with phosphoric acid at a flow rate of 2 ml min(-1) for cefadroxil. Quantitation was achieved with UV detection at 275 and 260 nm for cefuroxime and cefadroxil, respectively, based on peak area with linear calibration curves at the concentration ranges of 2-10 microg ml(-1) for cefuroxime and 5-20 microg ml(-1) for cefadroxil. The proposed methods were applied to the determination of dissolution rate for tablets and capsules containing each drug. The urinary excretion patterns as the cumulative amounts excreted have been calculated for each drug using the proposed methods.     

HPLC determination of valproic acid in human serum

A HPLC method for the determination of valproic acid (VA) in human serum using diazepam as internal standard (I.S.) is described. The eluates were separated with a C18 250 x 4.6 mm internal diameter reversed phase column maintained at a temperature of 50 degrees C. A mobile phase consisting of acetonitrile and 0.05 M phosphate buffer (pH 3.0) 45:55 v/v was used at a flow rate of 1.2 ml/min. Wavelength was switched from 360 nm to 210 nm during valproic acid retention. The method was linear over a concentration range of 20 to 160 microg/ml for valproic acid. Recovery was greater than 94% over a concentration range of 20 to 120 microg/ml and the limit of quantitation was 1 microg/ml. The intra day and inter day relative standard deviation (R.S.D.) measured at 20, 60, 80 and 120 microg/ml ranged from 1.46 to 5.34% and 0.83 to 5.03% respectively. The method is simple, rapid, accurate and sensitive and it was used for Therapeutic Drug Monitoring (TDM) in Indian epileptic patient population. The results obtained with this method correlated well with clinical practice.     

A simple and rapid high-performance liquid chromatographic method for the determination of bisoprolol fumarate and hydrochlorothiazide in a tablet dosage form

A simple and precise high performance liquid chromatographic method has been developed and validated for the simultaneous determination of bisoprolol fumarate (BF), and hydrochlorothiazide (HCTZ) in a tablet formulation. Chromatography was carried out at 25 degrees C on a 4.6 mm x 250 mm, 5 microm cyano column with the isocratic mobile phase of 0.1M aqueous phosphate buffer, acetonitrile and tetrahydrofuran (85:10:5, v/v/v) at a flow rate of 1.0 ml/min. The UV detection was carried out at 225 nm. HCTZ and BF were separated in less than 10 min with good resolution and minimal tailing, without interference of excipients. The method was validated according to ICH guidelines and the acceptance criteria for accuracy, precision, linearity, specificity and system suitability were met in all cases. The method was linear in the range of 50-150 microg/ml for BF and 125-375 microg/ml for HCTZ.     

HPLC determination of phenylpropanolamine in pharmaceutical preparations using 4-dimethylaminobenzaldehyde as a derivatizing reagent

A method is described for the high performance liquid chromatographic (HPLC) determination of phenylpropanolamine (PPA) based on precolumn derivatization with 4-dimethylaminobenzaldehyde (DAB) and elution from phenomenex C-18 column with methanol-water and detection by spectrophotometry at 418 nm. Linear calibration was obtained with 9.4--46.9 microg ml(-1) with a detection limit of 4.7 ng ml(-1). Vitamin B(12) and rifampicin when present together with PPA separated completely and could be determined simultaneously. PPA was determined in pharmaceutical preparations with a relative standard deviation of 0.6--1.6%.     

Method development and validation for the GC-FID assay of p-cymene in tea tree oil formulation

This paper describes the development and validation of an isothermal gas chromatography-flame ionisation detection (GC-FID) method for the assay of pure tea tree oil. The chromatographic conditions of the method employ a 5% carbowax packed column (20 m x 0.25 mm), isothermal elution with hydrogen at a column flow of 36 ml/min, injector and detector temperature at 220 degrees C and oven temperature at 100 degrees C, and a 1.5 microl injection volume. Samples and standard were diluted in hexane. The calibration curve for p-cymene was linear (r2=0.9995) from 20 to 120% range of the analytical concentration of 100 microg/ml. The precision of this method was calculated as the relative standard deviation (R.S.D.) was 0.66% (n=6). The R.S.D. for intermediate precision study was 0.13 and recovery of the p-cymene ranged between 93.39 and 97.86%. The limits of detection and quantitation were determined to be 2.08 and 10.39 ng/ml, respectively.     

HPLC assay and pharmacokinetic study of atorvastatin in beagle dogs after oral administration of atorvastatin self-microemulsifying drug delivery system

A specific and accurate reversed-phase HPLC with UV detection was developed for the assay of atorvastatin in beagle dog plasma. Indomethacin was used as the internal standard. Atorvastatin was extracted by protein precipitation, the extracts were injected into a Kromasil C8 column (150 mm x 4.6 mm, 5 microm) with UV wavelength set at 270 nm. The mobile phase consisted of acetonitrile:0.1 mol/L ammonium acetate buffer (pH 4.0) (65:35% v/v) at a flow rate of 1.0 ml/min. The column was at ambient temperature (25 degrees C). The injection volume was 25 microl. The blank plasma did not interfere with the determination of atorvastatin and indomethacin. A good linear relationship was obtained between the peak area ratio of atorvastatin to indomethacin and the concentration of atorvastatin over the range of 0.05 to 2.5 microg/mL. The limit of quantification was 25 ng/mL, the limit of detection was 8 ng/ml. The total chromatographic analysis time was within 9 min. The method is accurate, precise and fast for the assay of atorvastatin in plasma following oral administration of an atorvastatin SMEDDS to healthy beagle dogs.     

An improved and validated LC method for resolution of bicalutamide enantiomers using amylose tris-(3,5-dimethylphenylcarbamate) as a chiral stationary phase

An improved HPLC method for determination of enantiomeric purity of bicalutamide in drugs and pharmaceuticals was developed and validated. Baseline separation with resolution >/=6.0 was achieved within 10 min on Chiralpak AD-H (250 mm x 4.6 mm; particle size 5 microm) column using n-hexane:2-propanol (65:35 v/v) as mobile phase at a flow rate of 1.0 ml/min at 25 degrees C. The detection was made at 270 nm using UV detector while a polarimetric detector connected in series was used for identification of enantiomers. The effects of 2-propanol, ethanol and temperature on enantioselectivity and resolution of enantiomers were evaluated. The method was validated in terms of accuracy, precision and linearity in the range of 10-250 microg/ml and the r(2) was >0.9999. The recoveries were 99.68-100.25% with <1% R.S.D. The limits of detection (LOD) and quantification (LOQ) of enantiomers were (2.4, 3.0 and 7.6, 9.3) x 10(-8)g/ml for (S)-(+)-BCT and (R)-(-)-BCT enantiomers, respectively. The method was found to be suitable for rapid determination of enantiomeric purity of bicalutamide in bulk drugs and pharmaceutical formulations.     

Evaluation of monolithic C18 HPLC columns for the fast analysis of pilocarpine hydrochloride in the presence of its degradation products

Monolithic Performance C18 HPLC columns (Chromolith Performance RP-18e, Merck) were applied for the determination of pilocarpine hydrochloride in the presence of its degradation products isopilocarpine, pilocarpic acid and isopilocarpic acid. The method was validated using a set of six monolithic columns and compared to a conventional C18 column. The separation of pilocarpine from its degradation products was investigated on monolithic columns at different flow rates from 1 to 9 ml/min. Superior resolution was obtained using monolithic columns over the conventional C18 column at the same flow rate of 1 ml/min with a total run time of 9 min compared to 13.5 min for the conventional C18 column. Comparable resolution to that obtained in the C18 column (but with better peak symmetry) was obtained at a flow rate of 4 ml/min, although the total run time was reduced to 3.5 min. The precision for both retention time and peak area was investigated over a wide concentration range and found to be equal, or slightly better on Chromolith Performance compared to the conventional column. The overall RSDs% ranged from 0.5% to 1.16% for the conventional column, while for monolithic columns ranged from 0.38% to 0.87% at a flow rate of 1 ml/min and from 0.38% to 0.89% at a flow rate of 4 ml/min. Monolithic column to column reproducibility (n = 6) was measured. The RSDs% ranged from 0.34% to 0.68% for retention time and from 0.3% to 0.94% for peak areas. The detection and quantitation limits on monolithic columns at both flow rates (1 and 4 ml/min) were found to be 0.17 microg/ml and 0.5 microg/ml, compared to 0.31 microg/ml and 1 microg/ml on the conventional column. Monolithic silica rods have also shown the advantage of reducing the time to wash and to re-equilibrate the column. The method showed good linearity and recovery and was found to be suitable for the analysis of pilocarpine hydrochloride formulations.     

A sensitive method for the determination of gemfibrozil in human plasma samples by RP-LC.

A sensitive high-performance liquid chromatographic assay for the quantitative determination of gemfibrozil is described in this work. Ibuprofen was used as internal standard. The assay involved a single cyclohexane extraction and LC analysis with fluorescence detection. Chromatography was performed at 40 degrees C on a Hypersil ODS column. The mobile phase was a mixture of a solution of phosphoric acid 0.4% and acetonitrile (45:55). The method was validated. The detection limit of this method was 0.025 microg ml(-1); only 0.5 ml of the plasma sample was required for the determination. The calibration graph was linear from 0.05 to 0.5 microg ml(-1) and required a cubic equation from 0.5 to 30 microg ml(-1). Intra and inter-day precision (C.V.) did no exceed 15%. Mean recoveries were of 90.15+/-6.9% (C.V.'s<8%) for gemfibrozil and 93.10% for ibuprofen Applicability of the method was demonstrated by a pharmacokinetic study in normal volunteers who received gemfibrozil by oral route.     

Sensitive LC determination of ciprofloxacin in pharmaceutical preparations and biological fluids with fluorescence detection

A simple and highly sensitive isocratic reversed-phase high-performance liquid chromatographic method (RP-HPLC) has been developed for the determination of ciprofloxacin. Separation of ciprofloxacin and anthranilic acid (internal standard) was achieved on a Kromasil 100, C(18), 5 microm (250 x 4.6 mm i.d.) reversed-phase column, using fluorescence detection with lambda(exc)=300 nm and lambda(emi)=458 nm. The mobile phase consisted of acetonitrile-methanol-acetate buffer (pH 3.60; 0.05 M) (10:30:60 v/v/v) containing 1% v/v acetic acid. The analysis was performed in less than 9 min, with a flow rate of 0.8 ml min(-1). A rectilinear relationship was observed for concentrations between 0.005 and 1.0 microg ml(-1) of ciprofloxacin in aqueous standard solutions and serum and the detection limit was 20 pg injected on-column. The intra- and inter-day relative standard deviation (n=8) ranged from 1.6 to 2.6% and from 1.9 to 4.8%, respectively, calculated at three concentration levels of standard solutions. Direct measurements of ciprofloxacin in pharmaceutical preparations and in serum, after precipitation of proteins, were performed with high precision and accuracy. The application of the method to urine samples involved a solid-phase extraction treatment of the samples using C(18) cartridges. The linear working range in urine extended from 0.05 to 2.0 microg ml(-1) and the detection limit was 0.2 ng injected on-column.     

Stability of cefepime in aqueous eye drops

The aim of the studies was to determine HPLC the stability of cefepime in 1% and 5% buffered eye drops of developed formulary composition, which were stored for 30 days at the temperature of 4 degrees C and 20 degrees C, protected from light. Separation was performed on RP18 Gemini octadecylsilane column (250 mm x 4.6 mm, 5.0 microm) at a temperature of 25 degrees C. The mobile phase consisted of 0.015 M solution of sodium salt of pentane sulphonic acid brought to pH 4.0 with glacial acetic acid and 45% KOH solution and acetonitrile 94:6 w/w, with detection of 254 nm. The method was linear in the range of 12.6-125 microg/ml (R2 = 0.9996). The limit of detection (LOD) was 3 microg/ml and limit of quantification (LOQ) was 10 microg/ml. 10% degradation of cefepime in 1% and 5% buffered eye drops stored at the temperature of 4 degrees C, depending on the composition of the eye drops, occurred after 21-27 days in 1% eye drops and 18-21 days in 5% eye drops. In the eye drops, which were stored at the temperature of 20 degrees C, 10% degradation of cefepime took place on the third day of storage regardless of formulary composition of 1% and 5% drops. Cefepime stability lasting a couple of weeks in 1% and 5% solution allows extemporaneous preparation of buffered eye drops containing cefepime.     

Indirect polarographic and cathodic stripping voltammetric determination of cefaclor as an alkaline degradation product

Cefaclor is not reducible at a mercury electrode, but it can be determined polarographically and by cathodic stripping voltammetry as its initial alkaline degradation product which is obtained in high yield by hydrolysis of cefaclor in Britton-Robinson (B-R) buffer pH 10 at 50 degrees C for 30 min (reduction peak at pH 10, -0.70 V). Differential pulse polarographic calibration graphs are linear up to at least 1 x 10(-4) mol/l(-1). Recoveries of 93% of the cefaclor (n = 3) were obtained from urine spiked with 38.6 microg/ml(-1) using this polarographic method with 1 ml urine made up to 10 ml with pH 10 buffer. Using cathodic stripping voltammetry and accumulating at a hanging mercury drop electrode at - 0.2 V for 30 s, linear calibration graphs were obtained from 0.35 to 40 microg/ml(-1) cefaclor in B-R buffer pH 10. A relative standard deviation of 4.2% (eta = 5) was obtained, and the limit of detection was calculated to be 2.9 ng/ml(-1). Direct determination of cefaclor in human urine (1 ml of urine was made up to 10 ml with pH 10 buffer) spiked to 0.39 microg/ml(-1) was made (recovery 98.6%).     

Development and validation of a novel LC/ELSD method for the quantitation of gentamicin sulfate components in pharmaceuticals

The equivalent response of evaporative light scattering detector (ELSD) for compounds of similar structure is exploited to develop an LC/ELSD method for the simultaneous quantitation of the four main components of gentamicin sulfate, using as external standard the one main component kanamycin. A C18 column was used along with a mobile phase consisting of H2O (containing 35.4 microg/ml of trichloroacetic acid and 0.89 microl/ml of trifluoroacetic acid)-methanol-acetonitrile (990:5:5, v/v/v), in an isocratic mode at 1.1 ml/min. Parameters of ELSD were 50 degrees C for evaporation temperature and 3.0 bar for pressure of carrier gas (N2). A logarithmic calibration curve was obtained for sulfate (tR = 1.9 min) from 4.2 to 150 microg/ml (r > 0.994) with a precision of 0.18%R.S.D. Kanamycin and the four gentamicin components (C(1a), C2, C(2a) and C1) were eluted at 3.2, 4.6, 5.9, 7.1 and 8.7 min, respectively, with good resolution (Rs > 1.5). Logarithmic calibration curve was obtained for each component (r > 0.99) with statistically equal slopes varying from 2.457 to 2.558. The mass range of total gentamycin was 35-240 microg/ml. The proposed method was applied for the determination of gentamicin components and sulfate in raw materials and pharmaceutical formulations (injection, drops and cream) without any pretreatment except cream, for which liquid-liquid extraction was required. Recovery from standard addition experiments in commercial formulations was 99-100% regarding total gentamicin and 89-108% regarding individual components, with a precision (%RSD, n = 4) 0.7-5.8%.     

HPLC method for the determination of nystatin in saliva for application in clinical studies

An isocratic high-performance liquid chromatographic method was developed, optimized and validated for the determination of nystatin in human saliva (UV and fluorescence detection). A reversed-phase Luna C18 column (25 degrees C), with a mobile phase of MeOH, H2O, and DMF (70:20:10, v/v/v), and a flow-rate of 0.8 ml/min were used. The elution time for nystatin was 5.8+/-0.2 min. Calibration curves in human saliva were linear from 0.78 to 50 microg/ml. Limits of quantification were 0.78 microg/ml and 0.75 microg/ml for UV and fluorescence detection, respectively. The accuracy and precision values of intra- and inter-day variation studies were within acceptable limits, according to FDA guidelines. The described method has proved to be useful to give accurate measurements of nystatin in real samples.     

A simple HPLC method with pulsed EC detection for the analysis of creatine

The objective of this study was to develop a simple and sensitive LC method for the determination of creatine in aqueous solutions as well as in rat plasma using electrochemical detection. The chromatographic system consisted of a GP50 gradient pump, an ED40 pulsed electrochemical detector, and an AI-450 chromatography automation system (Dionex). The mobile phase consisted of a mixture of water, acetonitrile, 0.01 M sodium acetate, and 1.0 M sodium hydroxide (2.5:2.5:90:5, V/V/V/V) at a flow rate of 1.0 ml/min. The chromatographic separation was achieved at 45 degrees C on a column with a polyhydroxylated glucose and sulfonated stationary phase. The retention times of creatine and creatinine was 3.50 and 4.73 min, respectively, with creatine fully resolved from its major degradation product, creatinine. The standard curves were linear over the concentration range of 0-20 microg/ml. Within-day and day-to-day relative standard deviations (R.S.D.) were less than 10%. This method was used to study dissolution characteristics of various creatine salts in water.     

Quantitative determination of spinosin in rat plasma by liquid chromatography-tandem mass spectrometry method

A sensitive method for the quantitative determination of spinosin in rat plasma was developed and validated using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The analytes of interest were extracted from rat plasma samples by methyl tert-butyl ether (MTBE) after acidification with 1.0% acetic acid aqueous solution. Chromatographic separation was achieved on an Agilent Zorbax SB-C(18) (50 mm x 4.6 mm, 5 microm) using a isocratic mobile phase consisting of acetonitrile-water (30:70, v/v) with 1% isopropyl alcohol and 0.01% heptafluorobutyric acid. The flow rate was 0.2 ml/min. The column temperature was maintained at 25 degrees C. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via electrospray ionization (ESI). The calibration curve was linear over the range of 1.00-400 ng/ml in rat plasma, with 1.00 ng/ml of the lower limit of quantification (LLOQ). The inter- and intra-day precisions and accuracy for all samples were satisfactory. The validated method was successfully applied for the pharmacokinetic study of spinosin in rat. After oral administration of 20mg/kg spinosin to rats, the main pharmacokinetic parameters of T(max), C(max), T(0.5) and AUC(0-T) were 5.33+/-0.58 h, 132.2+/-10.6 ng/ml, 4.89+/-0.37 h, 1.02+/-0.09 microg h/l, respectively.     

Effect of system variables involved in packed column supercritical fluid chromatography of stavudine taken as model analyte using response surface methodology along with study of thermodynamic parameters

A multifactor optimization technique is successfully applied to study the effect of simultaneously varying the system variables on feasibility of stavudine analysis by packed column supercritical fluid chromatography (PC-SFC). The effect of simultaneously varying the pressure, temperature and modifier concentration was studied to optimize the method in order to obtain excellent chromatographic figures of merit. The method is based on isocratic elution using methanol-modified supercritical carbon dioxide as the mobile phase at the flow rate of 3.0 ml/min through a JASCO Finepak SIL-5, ODS [C(18) (5 microm, 25 cm x 4.6 mm, i.d.)] column support using photodiode array detection. The optimal conditions were determined with the aid of the response surface methodology using 3(3) factorial designs. From the response surface graphs optimum regions were selected to be +1, -1, and +1 for temperature (60 degrees C), pressure (20 MPa) and percent modifier concentration (17.81%, v/v), respectively. Linearity dynamic range was found to be in the range of 2.0-150.0 microg/ml with significantly high value of correlation coefficient. The method was validated for precision, robustness and recovery to assess the viability of the established method. The chromatographic limit of detection and quantitation were 0.80 and 1.50 microg/ml respectively. The method has been successfully used to analyze commercial dosage form to assess the chromatographic performance of SFC system which was found to be 99.91%+/-1.62. The present work briefs the thermodynamic applications of PC-SFC with an emphasis on the results of stavudine. The foremost of such applications is the determination of solute diffusion coefficient in supercritical mobile phase by Taylor-Aris peak broadening technique.