INTRODUCTION:

· A method of separating and identifying the components of a complex mixture by differential movement through a two-phase system, in which the movement is effected by a flow of a liquid or a gas (mobile phase) which percolates through an adsorbent (stationary phase) or a second liquid phase.¹

· The Russian botanist Mikhail Tswett first used the term ‘Chromatography’ (Latin for colored drawing’) in 1906, and was used to separated plant pigments¹

· High-performance liquid chromatography (HPLC) is the term used to describe liquid chromatography in which the liquid mobile phase is mechanically pumped through a column that contains the stationary phase. An HPLC instrument, therefore, consists of an injector, a pump, a column, and a detector.²

Figure1: high performance liquid chromatography

Types of Chromatography:

Paper chromatography:

This is probably the first, and the simplest, type of chromatography that people meet. A drop of a solution of a mixture of dyes or inks is placed on a piece of chromatography paper and allowed to dry. The mixture separates as the solvent front advances past the mixture.

Column Chromatography:

Column chromatography is a separation technique in which the stationary bed is within a tube. Differences in rates of movement through the medium are calculated to different retention times of the sample 3.

Thin Layer Chromatography:

Thin layer chromatography (TLC) is a widely employed laboratory technique and is similar to paper chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate.

Gas Chromatography:

Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC), is a separation technique in which the mobile phase is a gas. Gas chromatography is always carried out in a column, which is typically "packed" or "capillary".

Ion Exchange Chromatography:

In this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently attach anions or cations on to it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces.

Types of HPLC Techniques:

(A)Based on Modes of Chromatography:

(1) Normal-phase chromatography

(2) Reversed-phase chromatography (RPC)

(B) Based on Principle of Separation:

(1) Adsorption chromatography

(2) Size-exclusion (or) Gel Permeation chromatography

(3) Ion-exchange chromatography

(4) Affinity chromatography

(C) Elution Technique:

(1) Isocratic Separation

(2) Gradient Separation

(D) Based On Type Of Analysis:

(1) Qualitative Analysis

(2) Quantitative Analysis

New Trends in HPLC Technique:

HPLC is compared with the classical techniques are characterized by:

· Rapid Resolution Liquid chromatography (RRLC)

· Ultra Performance Liquid chromatography (UPLC)

· Ultra Fast Liquid chromatography (UFLC)

· Nano Liquid chromatography (Nano LC)

Rapid Resolution Liquid Chromatography (RRLC):

RRLC system was designed to provide highest analysis speed, resolution and pressure at a minimum. This analysis has become a routine method in the pharmaceutical industry. It holds excellent peak shapes, enhanced reproducibility, high sensitivity, high-speed detection with reduced analysis cost, and is valuable for the quality control of herbal medicine. The separation resolution and reduction of analysis time has continually improved in High Performance Liquid Chromatography (HPLC). Since then, HPLC using smaller particles has become more popular.⁴

Figure 2: Rapid Resolution Liquid Chromatography (RRLC)

Principle:

The underlying principles of this evolution are governed by the van Demeter equation, which is an empirical formula that describes The relationship between linear velocity (flow rate) and plate height (HETP or 1/column efficiency)

Applications of RRLC:

RRLC-tandem mass spectrometry method for the determination of endocrine disrupting chemicals (EDCs) examples: Bentazone, salicylic acid, silica gel pharmaceuticals and personal care products (PPCPs) in waste water irrigated soils. Analysis for quality control of Rhodiolarosea roots and commercial standardized products.

Ultra Performance Liquid Chromatography (UPLC):

UPLC refers to Ultra Performance Liquid Chromatography. It improves in three areas: chromatographic resolution, speed and sensitivity analysis. It uses fine particles and saves time and reduces solvent consumption.^5-8UPLC is comes from HPLC. HPLC has been the evolution of the packing materials used to effect the separation.

Figure 3: Graph of UPLC

Principle:

The UPLC is based on the principal of use of stationary phase consisting of particles less than 2 μm (while HPLC columns are typically filled with particles of 3 to 5 μm). The underlying principles of this evolution are governed by the van Deemter equation, which is an empirical formula that describes the relationship between linear velocity (flow rate) and plate height (HETP or column efficiency).

Applications of UPLC:

UPLC used in Identification of Metabolite, impurity Profiling Bio analysis / Bioequivalence studies Analysis of amino acids, natural products and traditional herbal medicine^.

Ultra Fast Liquid Chromatography (UFLC)

Figure 4: Diagram of UFLC

It is ten times higher speed and three times better separation than other LC techniques and offers outstanding speed and separation even at normal pressure levels. By maximizing the column and performance of the entire system UFLC minimizes the deviation from the van Demeter theory ⁹. The Prominence UFLC series provides ultrafast analysis, while maintaining high analytical precision and reliability¹⁰

Applications of UFLC:

It is used for analysis of Isoflavones, enables the use of high viscosity mobile phase and analysis under lower temperature.

Nano Liquid Chromatography (Nano Lc):

Some definitions have been found in the literature based on column diameter and mobile phase flow rates^11-13. Some workers defined NLC as chromatographic modality having mobile phase flow rate at nano ML per minute. But, the detection aspect of this chromatography which is very important in analytical science was not taken into consideration until then. Later in 2009, Ali et al gave an exact and scientific definition i.e. a modality of chromatography involving samples in nano liters, mobile phase flow rates in nano milli liter per minute, with detection at nano grams per milli liter.^14-15

Principle:

There is no agreement about the terminology of micro scale LC. The terms “microbore,” “micro column” and “capillary” LC are used interchangeably for micro columns of different i.d. (According to Cher vet et al., separations performed using columns of 0.50–1.0 mm i.d. are described as micro-LC; columns of 100–500 mm i.d. are described as capillary-LC; finally, separations using columns of 10–100 mmi.d. are described as

Figure 5: Diagram of Nano LC

Applications of Nano LC:

It is used in the analysis of peptides/proteins in proteomics, Biological and environmental samples Determine dyessotoxin for the determination of Yessotoxin in marine phytoplankton by Nano LC with hybrid quadruple time-off light mass spectrometry. Some workers defined NLC as chromatographic modality having mobile phase flow rate at nano ML per minute. But, the detection aspect of this chromatography which is very important in analytical science was not taken into consideration until then. Later in 2009, Ali et al gave an exact and scientific definition i.e. a modality of chromatography involving samples in nano liters, mobile phase flow rates in nano milli liter per minute, with detection at nano grams per milli liter.¹⁰

Instrumentation:

1. Pumps:

pumps the mobile phase at a specific flow rate in mL/min. The pump pressure is normally between 400-600 bar. Pumps ensure constant mobile phase flow.

2. Injector:

Introduces the sample into the column (about 5-20 ìL). Sampling multi-way valves with inner or outer loops are applied for injection.

3. Column:

Provides separation through high pressure created by the small particles. Analytical columns are stainless steel (most often) or glass tubes of inner diameter between 1 to 10 mm and length of 5 to 50 cm.

4. Detector:

Various detectors are available in LC. The most common is the spectrophotometric detector. It can work in both UV and visible regions of the spectrum; eventually at any moment of analysis the whole spectrum can be recorded DAD).

Applications:

The information that can be obtained using HPLC includes identification, quantification, and resolution of a compound.

Chemical Separations:

It is based on the fact that certain compounds have different migration rates given a particular column and mobile phase, the extent or degree of separation is mostly determined by the choice of stationary phase and mobile phase.

Purification:

Purification is defined as the process of separating or extracting the target compound from a mixture of compounds or contaminants. Each compound showed a characteristic peak under certain chromatographic conditions.

Identification:

Generally assay of compounds are carried using HPLC. The identifying peak should have a reasonable retention time and should be well separated from extraneous peaks at the detection levels which the assay will be performed.¹³

CONCLUSION:

HPLC is an assertive analytical technique with sophisticated technologies that have been extensively practiced from decades. RRLC offers improved run times and increased sensitivity over conventional HPLC based methods. We applied the fast RRLC technique to the analysis of general chemical compounds. Columns with small internal diameters and / or short column lengths are more susceptible to extra-column band-broadening for high speed separation in UPLC. Ultrafast analysis means a significant enhancement in sample throughput (5-10times) and productivity compared to a conventional HPLC. Nano LC is the latest innovation in separation science in which detections can achieved at nano gram or lower levels.

REFERENCES:

1. Ettre L S. M.S. Tswett and The invention of chromatography, LC-GC, 21, 2003, 458-467.

2. Narayudu Yandamuri Comparative Study of New Trends in HPLC: A Review International Journal of Pharmaceutical Sciences Review and Research Int. J. Pharm. Sci. Rev. Res., 2013; 23(2): 52-57.

3. Dr. S. Ravi sankar, Text book of Pharmaceutical analysis, Rx Publications, 3rd Edition, 2.22.5, 13.1, 17.14-17.18 & 18.2-18.6.

4. Maa YC, Wanga XQ, Houa FF, Ma J, Luoa M, Chena A, Jin P, Lua S, Xuaa I, Rapid resolution liquid chromatography (RRLC) analysis and studies on the stability of Shuang- Huang-Lian preparations. Journal of Pharmaceutical and Biomedical Analysis., 2011; 54: 2011, 265–272.

5. Dr. Michael E. Swartz and Brian J. Murphy, Ultra performance liquid chromatography: Tomorrow’s HPLC technology today, Novel Phases FOR HPLC, as published in LPI- June 2004. URL: www.labint-online.com/uploads/tx_ttproducts/ datasheet/ ultra-performance-liquid-chromatographytomorrow's-hplc-technology-today.pdf

6. Meenakshi D, Meenaxi M, A Review on Ultra Performance Liquid Chromatography, International Journal of Drug Development & Research, 5, 2013, 29-34.

7. Ashok Kumar, Gautam S, Anroop N and Rishbha S, UPLC: A Preeminent Technique in Pharmaceutical Analysis, Acta Poloniae Pharmaceutica ñ Drug Research, 69, 2012,371-380.

8. Swartz M. E. and Murphy B., Pharm. Formulation Quality, 6, 2004, 40

9. Ultra-fast liquid chromatography, URL:.http//Schimadzu.co.UK.

10. www.shimadzu.com/about/pressrelease/oh80jt000000 abel.html

11. Kenichirotanaka, Tadayuki Yamaguchi, Takeshi Goto, Hirohisa Mikami and Stanly M.lok, Available from: URL:www.ssi.shimadzu.com/about/pdfs/UF LC_Panax_Ginseng.pdf

12. Chervet J.P., Ursem M., Salzmann. J.P., Instrumental requirements for nano-scale liquid chromatography. Anal. Chem., 1996; 68: 1507-1512.

13. Saito.Y, Jinno.K, Greibrokk T. Capillary columns in liquid chromatography: Between conventional columns and microchips. J. Sep. Sci., 2004; 27: 1379-1390.

14. Hernandez-Borges J.H, Aturki Z, Rocco A, Fanali S. Recent applications in nano-liquid chromatography. J. Sep. Sci, 30, 2007, 1589-1610.

15. He Qing., Integrated nano liquid chromatography system on a chip., Ph.D. Thesis, California Institute of Technology, Pasadena, California, USA, June 21, 2005.

Received on 03.09.2018 Accepted on 21.10.2018

Asian J. Pharm. Ana. 2018; 8(4): 233-236.

DOI: 10.5958/2231-5675.2018.00042.X