14^th International Conference & Expo on Chromatography Techniques February 21-22, 2022 Berlin, Germany

Chromatography-HPLC is a popular method of analysis for natural products because of its high accuracy, precision and is not differed by the stability or the volatility of the compounds. HPLC combined with diode array detector (HPLC-DAD), mass spectrometer (HPLC-MS) have been successfully utilized for the qualitative and quantitative determination of various types of phyto-constituents like alkaloids, glycosides, tannins, tri-terpenes, flavonoids etc. HPLC methods are used readily for the determination of drug in biological fluids and pharmaceutical dosage forms. HPLC determination with spectroscopic detection is useful for routine quality control of drugs in pharmaceutical dosage forms and stability studies.

A chromatographic detector is capable of establishing both the identity and concentration of eluting components in the mobile phase stream. A broad range of detectors are available to meet different sample requirements. Detectors respond to a particular compound only and the response is independent of mobile phase composition and the response of bulk property detectors is dependent on collective changes in composition of sample and mobile phase. Specific detectors are UV-VIS, Photo diode array, fluorescence, and mass spectroscopic detectors. Bulk Property detectors include refractive index, electrochemical and light scattering detectors.

The hyphenated technique is developed from the coupling of a separation technique and an on-line spectroscopic detection technology. Several remarkable improvements in hyphenated analytical methods over the last two decades have significantly broadened their applications in the analysis of biomaterials, especially natural products, pre-isolation analyses of crude extracts or fraction from various natural sources, isolation and detection of natural products, chemical fingerprinting, testing of herbal products, de-replication of natural products, and metabolomics.

Rapid identification and characterization of known and new natural products directly from plant and marine sources without the necessity of isolation and purification can be achieved by various modern hyphenated techniques. Techniques like HPLC coupled to NMR (Nuclear Magnetic Resonance) or electrospray ionization tandem mass spectrometry (ESI-MS-MS) have been proven to be extremely powerful tools in natural product analysis, as they aid in the fast screening of crude natural product extracts or fractions for detailed information about metabolic profiles, with minimum quantity of material. Hyphenated HPLC techniques include HPLC-MS, HPLC-ESI-MS, HPLC-IC-MS, HPLC-NMR-MS, HPLC-DAD, HPLC-CE-MS, HPLC-UV, Coupling LC and MALDI-TOF.

Liquid chromatography is a fundamental separation technique in the life sciences and related fields of chemistry. Unlike gas chromatography, which is unsuitable for non-volatile and thermally fragile molecules, liquid chromatography can safely separate a very wide range of organic compounds, from small-molecule drug metabolites to peptides and proteins. Traditional detectors for liquid chromatography include refractive index, electrochemical, fluorescence, and ultraviolet-visible (UV-Vis) detectors. Some of these generate two-dimensional data; that is, data representing signal strength as a function of time. Others, including fluorescence and diode array UV-Vis detectors, generate three dimensional data. Three-dimensional data include not only signal strength but spectral data for each point in time.

Mass spectrometers also generate three-dimensional data. In addition to signal strength, they generate mass spectral data that can provide valuable information about the molecular weight, structure, identity, quantity, and purity of a sample. Mass spectral data add specificity that increases confidence in the results of both qualitative and quantitative analyses.

Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis.  Four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion. Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography. Chromatographic separation methods include adsorption chromatography, ion exchange chromatography, affinity chromatography, and size exclusion chromatography.

Chromatography and spectroscopy are orthogonal techniques, i.e. their types of information are very different and are specific. Chromatography is a separation method and spectroscopy is a technique which yields a ‘fingerprint’ of individual or from mixture of molecules. HPLC is a technique for separation, quantification and identification of components in a mixture. It is especially suitable for compounds which are not easily volatilized, thermally unstable and have high molecular weights. The advantage of UV method over HPLC method is that the UV method does not require the elaborate treatment and procedures usually associated with chromatographic method. It is less time consuming and economical. The HPLC and UV spectrometry methods are adequate methods to quantify a drug in pure form and its dosage form. Since these methods are simple, specific, rapid, precise and accurate, they may be successfully and conveniently adopted for routine quality control analysis of drugs in bulk and pharmaceutical dosage form.

The advancements in bio analysis, method development and validation reports, Micro and Nano technologies in bio analysis. Advance research stream mainly focuses on the combination of chemical functions using various patterning or immobilization techniques, and fusion with Nano-scale materials or molecules describes conventional micro analytical techniques like capillary electrophoresis, flow injection analysis, and micro electrodes.

Analytical method development and validation plays a major role within the event, manufacture and discovery of pharmaceuticals. Pharmaceutical products manufactured with over one drug, are referred as combination products, and are intended to satisfy previously unmet patients needed by combining the therapeutic effects of two or more drugs in one product. These combination products can exhibit overwhelming difficulties to the analytical chemist responsible of the event and validation of analytical methods.

Fingerprinting is a quality control model that builds upon spectroscopic and chromatographic technology. It is different from the traditional quality control model in the sense that fingerprinting looks at the “complete information” or comprehensiveness of the chromatograph, and displays integrated quality information. Since the secondary metabolites, which are chemical components of medicinal herbs, are inherently unstable, the fingerprints of these chemicals possess a fuzziness that cannot be precisely measured, just like the fuzzy phenomenon in our daily lives. Comprehensiveness and fuzziness are the two basic traits of a fingerprint. The similarity of fingerprints is established through these basic traits. Fingerprint analysis focuses on accurate identification (of similar peaks), and not on precise calculation. The comparison of fingerprints emphasizes similarity and the fingerprints compared do not need to be exactly the same. When it is impossible to find out all the complex components of a traditional medicine, fingerprints can be used to check the stability of the intrinsic quality of the medicine.

HPLC techniques are applied for purification and separation of various biological samples. The analysed samples are subjected to sequencing studies either manually or using different software’s. This is studied as Data mining and sequence analysis. HPLC is also used for characterization of various metabolites.

Chromatography-HPLC is the most versatile of all chromatography methods but also the most complex. It was first made available in the laboratory during the 1970s and is currently used for the analysis of amino acids, peptides, proteins, carbohydrates, lipids, nucleic acids and related compounds, vitamins, hormones, metabolites, and drugs. HPLC can be coupled to various detectors such as UV, fluorescence or mass spectrometry (LC/MS and LC/MS/MS) and is routinely used for quantitative analysis in biological samples such as blood, urine and other body fluids. HPLC consists of using a liquid mobile phase to pass under high pressure a mixture of analytes extracted from the sample through a column containing the stationary phase. Analyte separation is based on differences in interaction with both the mobile phase and the stationary phase.

HPLC is a proven method for isolating analytes of interest in complex matrices such as biological fluids. Its use in the clinical laboratory has steadily increased over the past decades as its unmatched analytical performance and versatility allows for testing of many different types of clinically relevant analytes. With the recent advances in detection technology such as mass spectrometry and sample preparation techniques such as bio-affinity chromatography and online automation, HPLC based methods will likely remain the gold standard of clinical testing for many of the current but also future biomarkers and therapeutic drugs.

Chromatography can be used at various stages of the food chain from determining the quality of food to detecting additives, pesticides and other harmful contaminants. Gas Chromatography – Mass Spectrometry (GC-MS) is a widely used technique for qualitative and quantitative analysis of food composition, food additives, flavour and aroma components and contaminants such as pesticides, natural toxins, veterinary drugs and packaging material. In recent years, to protect the health of consumers, more meticulous monitoring of food, more rigorous regulations with lower limits of quantification (LOQs) are required.

The use of liquid chromatography tandem mass spectrometry (LC/MSMS) is widely recognized as the extremely sensitive and highly specific technique of choice for the determination of food contaminants at trace levels including pesticides, veterinary drugs, natural toxins, and so-called “emerging contaminants”.

HPLC is widely used throughout the biological sciences. It is used by biochemists to purify peptides and proteins and used by molecular biologists to isolate nucleic acids, oligonucleotides and plasmids. It is also widely used in the biotechnology fields. For most biological samples, reverse-phase HPLC is used. Reverse-phase HPLC consists of a polar mobile phase and an a polar stationary phase.

Biological macromolecules can be either polar on non-polar dependent upon the side chain groups. For biological molecules that contain surface charges or polar side chains, there are many points for intermolecular attractions to form, be it by hydrogen bonding, hydrophilic/hydrophobic interactions etc. Because of this, these biological molecules are better suited for dissolution in polar solvents and will stay longer in the mobile phase. Whereas apolar molecules will prefer to adhere to the apolar stationary phase by van der Waals and dispersion interactions.