Two-dimensional liquid chromatography with reversed phase in both dimensions: A review.

Two-dimensional liquid chromatography (2D-LC), and in particular comprehensive two-dimensional liquid chromatography (LC×LC), offers increased peak capacity, resolution and selectivity compared to one-dimensional liquid chromatography. It is commonly accepted that the technique produces the best results when the separation mechanisms in the two dimensions are completely orthogonal; however, the use of similar separation mechanisms in both dimensions has been gaining popularity as it helps avoid difficulties related to mobile phase incompatibility and poor column efficiency. The remarkable advantages of using reversed phase in both dimensions (RPLC×RPLC) over other separation mechanisms made it a promising technique in the separation of complex samples. This review discusses some physical and practical considerations in method development for 2D-LC involving the use of RP in both dimensions. In addition, an extensive overview is presented of different applications that relied on RPLC×RPLC and 2D-LC with reversed phase column combinations to separate components of complex samples in different fields including food analysis, natural product analysis, environmental analysis, proteomics, lipidomics and metabolomics.

Green comprehensive two-dimensional liquid chromatography (LC × LC) for the analysis of phenolic compounds in grape juices and wine.

Grape juices and wines are rich in numerous groups of polyphenolic compounds which require a dedicated separation technique for such complex samples. LC × LC is considered the best technique for the analysis of such samples as it can achieve better resolution and higher peak capacity compared to 1D LC. The ever-growing demand for protecting the environment necessitates reducing or eliminating hazardous solvents to improve the environmental friendliness of analytical procedures. In this study, propylene carbonate was used as an eco-friendly mobile phase component in comprehensive two-dimensional liquid chromatography to analyze phenolic compounds in grape juices and a dealcoholized wine sample. Novel green RPLC × RPLC-DAD and RPLC × RPLC-MS methods were developed for the first time to identify phenolic compounds in five samples (two red grape juice samples, two white grape juice samples, and one dealcoholized wine sample). Four different RPLC × RPLC systems were developed; three systems were connected to a diode array detector (RPLC × RPLC-DAD), while the fourth system was connected to DAD and MS detectors (RPLC × RPLC-DAD-ESI-MS). Solvent X (propylene carbonate:ethanol, 60:40) was adopted as a green organic modifier in the first dimension (1D) and methanol in the second dimension (2D). The practical peak capacity and the surface coverage were calculated as metrics to measure the separation performance of all proposed systems. The orthogonality values for the setups ranged from 0.64 to 0.92 when calculated by the convex hull method, and from 0.54 to 0.80 when calculated by the asterisk equations method. The practical peak capacity production rate ranged from 14.58 to 22.52 peaks/min. The results revealed that the phenolic compounds were separated efficiently with good coverage of the 2D separation space and high peak capacity. A total of 70 phenolic compounds were detected based on MS data and information from the literature.

Parallel gradients in comprehensive multidimensional liquid chromatography enhance utilization of the separation space and the degree of orthogonality when the separation mechanisms are correlated.

Comprehensive two-dimensional liquid chromatography (LC×LC) offers increased peak capacity, resolution and selectivity compared to one-dimensional liquid chromatography. It is commonly accepted that the technique produces the best results when the separation mechanisms in the two dimensions are completely orthogonal, which necessitates the use of gradient elution for each second-dimension fraction. Recently, the use of similar separation mechanisms in both dimensions has been gaining popularity, but full or shifted gradients are still used for each second dimension fraction. Herein, we argue that when the separation mechanisms are correlated in the two dimensions, the best results can be obtained with the use of parallel gradients in the second dimension, which makes the technique nearly as user-friendly as comprehensive two-dimensional gas chromatography. This has been illustrated through the separation of a mixture of 39 pharmaceutical compounds using reversed phase in both dimensions. Different selectivity in the second dimension was obtained through the use of different stationary phase chemistries and/or mobile phase organic modifiers. The best coverage of the separation space was obtained when parallel gradients were applied in both dimensions, and the same was true for practical peak capacity.

Green Approaches to Sample Preparation Based on Extraction Techniques.

Preparing a sample for analysis is a crucial step of many analytical procedures. The goal of sample preparation is to provide a representative, homogenous sample that is free of interferences and compatible with the intended analytical method. Green approaches to sample preparation require that the consumption of hazardous organic solvents and energy be minimized or even eliminated in the analytical process. While no sample preparation is clearly the most environmentally friendly approach, complete elimination of this step is not always practical. In such cases, the extraction techniques which use low amounts of solvents or no solvents are considered ideal alternatives. This paper presents an overview of green extraction procedures and sample preparation methodologies, briefly introduces their theoretical principles, and describes the recent developments in food, pharmaceutical, environmental and bioanalytical chemistry applications.

Development of spectrofluorimetric method for determination of certain aminoglycoside drugs in dosage forms and human plasma through condensation with ninhydrin and phenyl acetaldehyde.

A simple and sensitive spectrofluorimetric method has been developed and validated for determination of amikacin sulfate, neomycin sulfate and tobramycin in pure forms, pharmaceutical formulations and human plasma. The method was based on condensation reaction of cited drugs with ninhydrin and phenylacetaldehyde in buffered medium (pH 6) resulting in formation of fluorescent products which exhibit excitation and emission maxima at 395 and 470nm, respectively. The different experimental parameters affecting the development and stability of the reaction products were carefully studied and optimized. The calibration plots were constructed with good correlation coefficients (0.9993 for tobramycin and 0.9996 for both neomycin and amikacin). The proposed method was successfully applied for the analysis of cited drugs in dosage forms with high accuracy (98.33-101.7)±(0.80-1.26)%. The results show an excellent agreement with the reference method, indicating no significant difference in accuracy and precision. Due to its high sensitivity, the proposed method was applied successfully for determination of amikacin in real human plasma.

Highly sensitive spectrofluorimetric method for determination of certain aminoglycosides in pharmaceutical formulations and human plasma.

A simple, reliable, highly sensitive and selective spectrofluorimetric method has been developed for determination of certain aminoglycosides namely amikacin sulfate, tobramycin, neomycin sulfate, gentamicin sulfate, kanamycin sulfate and streptomycin sulfate. The method is based on the formation of a charge transfer complexes between these drugs and safranin in buffer solution of pH 8. The formed complexes were quantitatively extracted with chloroform under the optimized experimental conditions. These complexes showed an excitation maxima at 519-524 nm and emission maxima at 545-570 nm. The calibration plots were constructed over the range of 4-60 pg mL(-1) for amikacin, 4-50 pg mL(-1) for gentamicin, neomycin and kanamycin, 4-40 pg mL(-1) for streptomycin and 5-50 pg mL(-1) for tobramycin. The proposed method was successfully applied to the analysis of the cited drugs in dosage forms. The proposed method was validated according to ICH and USP guidelines with respect to specificity, linearity, accuracy, precision and robustness. The high sensitivity of the proposed method allowed determination of amikacin and gentamicin in spiked and real human plasma.