Chromatography is a non-destructive procedure for resolving a complex mixture into its individual fractions or compounds. Chromatography is based on differential migration of solutes with the solvents. The solutes in a mobile phase go through a stationary phase. Those solutes with a high affinity for the mobile phase will spend more time in this phase than the solutes that prefer the stationary phase. As the solutes rise up through the stationary phase they separate, the process called chromatographic development. The fraction with greater affinity to stationary layer travels slower and shorter distance while that with less affinity travels faster and longer.

In normal-phase chromatography, the stationary phase is polar and the mobile phase is nonpolar. In reversed phase the stationary phase is nonpolar and the mobile phase is polar. Flash Column Chromatography (FCC) or Flash Chromatography is a quick and easy way to separate complex mixtures of compounds. FCC uses compressed air to push the solvent through the column. This helps provide better separation and reduces the amount of time required to run a column. Ion exchange chromatography (IEC) uses an ion exchange mechanism to separate analytes based on their respective charges. It is performed in columns but can also be useful in planar mode. IEC uses a charged stationary phase to separate charged compounds including cations, anions, amino acids, proteins and peptides. Conventional methods use stationary phase having an ion exchange resin that carries charged functional groups that interact with oppositely charged groups of the compound to retain. IEC is commonly used to purify proteins using fast protein liquid chromatography (FPLC).

Size-exclusion chromatography (SEC) is also known as gel permeation chromatography (GPC) or gel filtration chromatography and separates molecules according to their size or more accurately according to their hydrodynamic diameter or hydrodynamic volume. Small molecules enter the pores of the media where they are trapped and removed from the flow of the mobile phase. Mean residence time in the pores depends upon the effective size of the analyte molecules. Molecules that are larger than the average pore size of the packing are excluded and thus suffer essentially no retention; such species are the first to be eluted. It is a low-resolution chromatography technique and thus it is often reserved for polishing step of purification and is also useful for determining the tertiary structure and quaternary structure of purified proteins as can be carried out under native solution conditions.

Affinity chromatography is based on selective non-covalent interaction between an analyte and sample molecules. It is highly specific, but not robust. It is used in biochemistry in the purification of proteins bound to tags. Fusion proteins are labelled with compounds such as His-tags, antigens or biotin which bind to the stationary phase specifically. Later after purification, some of these tags are removed and the pure protein is obtained. Affinity chromatography utilizes a biomolecule's affinity for a metal (Zn, Cu, Fe, etc.). Columns are often manually prepared. Earlier affinity columns are used as a preparative step to flush out unwanted biomolecules. Some HPLC techniques utilize the properties of affinity chromatography. IMAC- Immobilized Metal Affinity Chromatography is used to separate aforementioned molecules based on the relative affinity for the metal. Often these columns can be loaded with different metals to create a column with a targeted affinity.

Chiral chromatography includes the separation of stereoisomers. Enantiomers have no chemical or physical differences apart from being three-dimensional mirror images. Traditional chromatography or other separation processes are incapable of separating these. For chiral separations to happen, either the mobile phase or the stationary phase must themselves be made chiral by inducing differing affinities between the analytes.

HPLC is a popular method of analysis for herbal products because of its accuracy, precision and not limited by the volatility or stability of the sample compounds. HPLC combined with diode array detector (HPLC-DAD), mass spectrometer (HPLC-MS) have been successfully employed in the qualitative and quantitative determination of various types of phytoconstituents like flavonoids, alkaloids, tannins, triterpenes, glycosides, sterols etc. HPLC methods are used in 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. Several synthetic molecules also are successfully evaluated both qualitatively and quantitatively. Pharmaceutically important antiviral drugs (lamivudine, stavudine, nevirapine, zidovudine), NSAIDs (acetaminophen, meloxicam, paracetamol, phenylbutazone,), antibiotics (amoxicillin, cefepime, rifampicin), CNS stimulants (amphetamine), betablockers (atenolol), preservatives (benzalkoniumchloride), Diuretics (hydrochlorthiazide), antidiabetics (metformin), anticancer (methotrexate), corticosteroids (prednisone, betamethasone, dexamethasone), anti-allergic (cetrizine), antidepressant (Nitroxazepine, milnacipran), veterinary anaesthetic (ketamine) , anti-inflammatory (sodium osagrel), secretolytic (ambroxol) and several other category drugs were successfully evaluated via HPLC.

HPLC columns are usually packed with porous, or pellicular particles. Pellicular particles are made from polymers, or glass. Pellicular particles are surrounded by a thin uniform layer of silica, synthetic resins, alumina, or an of ion-exchange resin. Partition HPLC uses liquid bonded phase columns. The liquid stationary phase is chemically bonded to the packing material. The packing material is usually hydrolysed silica which reacts with the bond-phase coatings like siloxanes. 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, chemotaxonomics, chemical fingerprinting, testing of herbal products, dereplication 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. The application of various hyphenated techniques even allows the discovery of new molecules, including complete and conclusive structure elucidation, and relative configurations as compared to time-consuming and costly isolation and purification processes. Hyphenated HPLC techniques include HPLC-UV, HPLC-DAD, HPLC-MS, HPLC-ESI-MS, HPLC-IC-MS, HPLC-NMR-MS, HPLC-CE-MS, Coupling LC and MALDI-TOF. MALDI (Matrix Assisted Laser Desorption Ionization) is a very sensitive technique for determining the mass of proteins, polymers and peptides. MALDI allows protein identification. MALDI sample preparation is fast and easy and therefore a primary choice in proteomics. Proteins, peptides, and polymers are fragile and tend to fragment when ionized by other ionization techniques. MALDI is attached to a time of flight (TOF) analyser which measures time it takes for the molecules to travel a fixed distance. MALDI uses a short laser pulse, instead of continuous laser making it a soft ionization technique.

The concept of on-chip chromatography includes a micro fabricated separation device. The availability of the fused-silica capillary marked such a significant breakthrough for GC that by 1989 more than 60% of all gas chromatographs manufactured were equipped to use fused silica capillary columns. Fused-silica capillaries also contributed to the developments of other micro separation technologies like supercritical fluid chromatography, capillary LC and CE. The success of one separation technique relies on sample introduction technologies, Separation column and sensitive detectors that can preserve chromatographic fidelity of high resolution chromatographic peaks, as is evidenced by the many injectors and detectors optimized and available for open tubular GC. A particle packed column is comprised of a nanolitre enrichment column and a micron or sub-micron separation column which is packed with suitable grade of C18. The HPLC-Chip is made from a biocompatible polyimide and the functionality of this chip is equivalent to conventional nanospray LC/MS.  Monoliths consist of a single rod of porous material with several unique features in terms of permeability and efficiency. Microfabricated column based on pillar-arrays were formed by arrays of nonporous silicon pillars with a diameter of approximately 4.3μm. The pillars were covalenty coated with a monolayer of hydrophobic C8-chains to enable reversed-phase LC separations.

UHPLC (Ultra-HPLC) or UPLC (Ultra Performance Liquid Chromatography)  is now being adopted in industrial labs, especially the pharmaceutical industry due to its high speed, high resolution and solvent saving.  A UHPLC method using a sub-2m column could reduce the analysis time by up to 80% and save the mobile phase consumption by at least 80% compared with an HPLC method using a conventional 3.5m column without sacrificing separation performance. In addition, the much shorter run time significantly reduces UHPLC method development scouting time. HPLC method development principles can be applied to UHPLC method development. Existing HPLC methods can be converted to UHPLC methods. Micro and Nano HPLC ensure high levels of flow rate flexibility and reproducibility for discovery and targeted proteomics. Hydrophilic interaction chromatography or hydrophilic interaction liquid chromatography (HILIC) is a variant of normal phase liquid chromatography that partly overlaps with other chromatographic applications such as ion chromatography and reversed phase liquid chromatography. It uses hydrophilic stationary phases with reversed-phase type eluents. Hydrophilic interaction liquid chromatography (HILIC) provides an alternative approach to effectively separate small polar compounds on polar stationary phases.

Quality can be designed to processes through systematic implementation of an optimization strategy to establish a thorough understanding of the response of the system quality to given variables, and the use of control strategies to ensure quality. Recently the FDA has begun to advocate the QbD (Quality by Design) methodology for the pharmaceutical sector. The concept of method development includes modeling of the influence of values of variables on quality, design of experiments, and simplification of processes as information is collected. The extension of QbD philosophies is now applied to the development of manufacturing processes and analytical methods. The ability of a chromatographic method to successfully separate, identify and quantitate species is determined by a powerful factor called experimental design which gives a powerful suite of statistical methodology. Automation of a process is one of the keys for increasing the productivity of a research group. Scaling-up a compound separation performed on an analytical system to a preparative liquid chromatography system requires an optimization step on the analytical column. This step concerns the development of the gradient method for the isolation of the target compound with the best balance between its purity, data throughput, and analysis time.

Chemometric Optimization is used to develop models which can be used to predict properties of interest based on measured properties of the chemical system. Chemometrics is applied to solve both descriptive and predictive problems in experimental natural sciences and chemistry. In descriptive applications, the analytical properties of chemical systems are modeled to understand the underlying relationships and structure of the system. Predictive applications include modelling of chemical systems to predict new properties. The datasets are very large and highly complex, involving thousands of variables, cases or observations. Chemometrics are applied for method development and validation in chromatography. Also data interpretation can be very close when such methods are applied.

HPLC can be used in both qualitative and quantitative applications that are for both compound quantification and identification. Normal phase HPLC is rarely used now, almost all HPLC separation can be performed in reverse phase. Reverse phase HPLC (RPLC) is ineffective in for only a few separation types. HPLC is applied for molecular weight determination, in analytical chemistry, pharmaceutical and drug science, clinical sciences, food technology, and consumer products, combinatorial chemistry, polymer chemistry, environmental chemistry and green chemistry.

HPLC is a very common method for metabolomic analysis. With the advent of electrospray ionization, HPLC is coupled to MS. HPLC has lower chromatographic resolution, requires no derivitization for polar molecules and separates molecules in the liquid phase. HPLC has the advantage of much wider range of analytes measurements with a higher sensitivity than GC methods. Relevant to proteomics, due to the complex structure and nature of proteins, instrumentation and methods development for sample cleanup, preconcentration, fractionation, chromatographic separation and detection becomes an immediate requirement for the identification of peptides and proteins. Latest techniques and equipment for separation and detection include nano-HPLC and multidimensional HPLC for protein and peptide separation. Relevant to genomics, HPLC is considered as most reliable and most sensitive technique to determine DNA methylation. The nucleotides and nucleosides of DNA are separated and quantified by HPLC-UV method. HPLC finds applications in glycomics and lipidomics where glycan part is cleaved either enzymatically or chemically from the target and subjected to analysis. In case of glycolipids, they can be analyzed directly without separation of the lipid component. HPLC is extensively used in lipidomics to separate lipids prior to mass spectrometry. Separation can be achieved by either normal-phase (NP) HPLC or reverse-phase (RP) HPLC.  For, untargeted lipidomic studies both RP and NP or Hydrophilic Interaction Liquid Chromatrography (HILIC) columns are used for increased coverage of lipidome. HPLC/UPLC separation of lipids may either be performed offline or online where the eluate is integrated with the ionization source of a mass spectrometer.

Robust HPLC techniques are applied for separation and purification of various biological samples. These analysed ones are subjected to sequencing studies either manually or using various softwares. This is studied as Data mining and sequence analysis with HPLC. HPLC is also used for characterization of various metabolites. HPLC also has relevant applications in Immunoinformatics, Molecular Modelling and Cheminformatics.