Short Review on Comparative Study of Chromatography

 

Nachiket V. Rajput*, Manvir V. Rajput, Vikas V. Patil, Pankaj S. Patil, Amol R. Pawar

Department of Pharmaceutical Quality Assurance,

Kisan Vidya Prasarak Sanstha’s Institute of Pharmaceutical Education, Boradi 425428

 

ABSTRACT:

Chromatography is a technique which is used for the separation of constituents in a mixture. HPLC is an advanced technique of column liquid chromatography. This article represents a short review of HPLC along with its principle and instrumentation. It describes about new trends in HPLC such as RRLC, UPLC, UFLC and Nano LC. Recent developments in chromatographic supports and instrumentation for liquid chromatography (LC) are enabling rapid and highly efficient separations. This new category of analytical separation science retains the practicality and principles of HPLC while increasing the overall interrelated attributes of speed, sensitivity and resolution. Today’s pharmaceutical industries are looking for new ways to cut cost and shorten time for development of drugs while at the same time improving the quality of their products and analytical laboratories are not exception in this trend. New techniques have mainly increased the resolution power for complex sample analysis.

 

 

 

INTRODUCTION:

The chromatography term is derived from the greek words namely chroma (colour) and graphein (to write). Chromatography is defined as a set of techniques which is used for the separation of constituents in a mixture. This technique involves 2 phases stationary and mobile phases. The separation of constituents is based on the difference between partition coefficients of the two phases. The chromatography is very popular technique and it is mostly used analytically.^1,2

 

In these chromatography techniques, HPLC is one of the chromatographic techniques, which is mostly used analytical technique. In This method stationary phase can be a liquid or a solid phase.

 

HPLC utilizes a liquid mobile phase to separate the components of a mixture. 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.³ High performance liquid chromatography (HPLC) has proven to one of the most and predominant technology used in analytical laboratories for the analysis of drugs worldwide during the past 30- plus years.^4,5

 

New Amendments 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)

 

Fig. 1 HPLC flow diagram

 

Rapid Resolution Liquid chromatography (RRLC):

The RRLC system was created to deliver the fastest analytical speed, best resolution, and lowest pressure. The world's fastest, most efficient, most adaptable LC system. It's become a popular method for increasing throughput, improving sensitivity, and lowering expenses. The RRLC system allows for faster analysis (theoretically up to 20x) while preserving the same resolution as traditional HPLC. Sub-2 micron column particle chemistry and high flow rates are used to achieve this. To reduce system back-pressure, higher temperatures are frequently used. With the extensive use of RRLC, the issue of HPLC detector compatibility has arisen.^6,7

 

RRLC system was designed to provide highest analysis speed, resolution and pressure at a minimum. 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. The RRLC system was designed to deliver the fastest analytical speed, best resolution, and at lowest pressure. In High Performance Liquid Chromatography, separation resolution and analysis time have continually increased (HPLC). Since then, HPLC using smaller particles has become more popular. In the pharmaceutical industry, this analysis has become standard practise. 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 medicines. This, along with other methodical experiments, resulted in the high-throughput, high-resolution HPLC that we know today. The usage of a lower column length results in a reduction in analysis time.⁸ A shorter column, on the other hand, may result in a loss of theoretical plates, lowering the chromatographic resolution necessary for a complex mixture of chemicals. The use of smaller size particles to compensate for the potential loss of resolution has resulted in more efficient columns. Long columns packed with smaller particles result in improved efficiency and resolution; using modern RRLC technology, analysis time can be cut in half without sacrificing chromatographic resolution.^9–11

 

Ultra-Performance Liquid chromatography (UPLC):

Ultra-Performance Liquid Chromatography (UPLC) takes the advantage of technological strides made in particle chemistry performance, system optimization, detector design and data processing and control. Using sub 2 mm particles and mobile phases at higher linear velocities and instrumentation that operates at higher pressures than those used in HPLC, dramatic increases in resolution, sensitivity and speed of analysis can be obtained. This new category of analytical separation science retains the practicality and principles of HPLC while creating a step function improvement in chromatographic performance. This review introduces the theory of UPLC and summarizes some of the most recent work in the field.^12,13

 

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. UPLC comes from HPLC. The packing materials used to effect separation have evolved into HPLC. An basic concept of HPLC states that when the particle size of the column packing decreases, the efficiency and consequently resolution increases. According to the common Van Deemter equation, there is a significant gain in efficiency as particle size shrinks to less than 2.5m, and it does not reduce with greater linear velocities or flow rates. Ultra-Performance refers to the ability to push speed and peak capacity to new heights by employing tiny particles. Elevated temperature chromatography also allows higher flow rates by lowering the viscosity of mobile phase which significantly reduces the column backpressure. However due to limited availability of packing materials stable at temperatures >100°C, method development using elevated temperature chromatography are not yet considered routine.^14,15

 

Ultra-Fast Liquid chromatography (UFLC):

It has ten times the speed and three times the separation of existing LC techniques, and it performs admirably even at normal pressure levels. UFLC minimizes departure from the van Demeter theory by optimizing the column and overall system performance. The Prominence UFLC series offers ultrafast analysis with high analytical precision and consistency.¹⁶

 

The simplest approach in the UFLC that for reduce the analysis time the short column (3-5cm) and high flow rates are use. The column is packed with particles having 3 to 3.5um in diameter. In 1998, Mutton reported a gradient separation of a text mixture transferred from a conventional column (4.6 x 150mm) to a short column (4.6 x 33mm), both packed with 3um particles. The initial method tool 40 min and was reduced to only 3.5 min in the shorter column, with a concomitant increase in the flow rate from 1 to 2.5ml/min. In the UFLC columns are packed with the porous particles instead use of monolith. Monolithic rods, made of silica or polymeric material, can accept high flow rates in conventional column length without generating high backpressures. Moreover, efficiency and resolution are comparable to silica particles of ca. 3um.¹⁷

 

Nano Liquid Chromatography (NANO LC):

Nano LC is defined as a chromatographic modality with a mobile phase flow rate of nano ml per minute. A chromatography mode involves nanoliter samples, nano milliliter per minute mobile phase flow rates, and detection at nanograms per milliliter. Because this type of modality is typically performed in microchips, it is also known as lab on chip chromatography.¹⁸

 

Nano-LC is an alternative to standard LC that offers more chemical analysis choices. Virtually all samples analyzed by conventional LC can be analyzed by a miniaturized technique. Capillary electrophoresis and capillary electrochromatography, as miniature liquid phase separations, complement and compete with nano-LC in this context. separations performed using columns of 0.50–1.0 mm i.d. are described as micro-LC; columns of 100–500mm i.d. are described as capillary-LC; finally, separations using columns of 10–100mm i.d. are described as nano-LC. This classification includes separations in microchips because nano-HPLC columns on chips have 20 to 100 mm as the i.d.¹⁹

 

Traditionally, packed high pressure LC analysis is performed in columns with an internal diameter (ID) of 4.6 mm. These so called analytical columns have typical flow rates of 1ml/min. Narrow bore columns have IDs of 1 and 2mm, while microcapillary columns have IDs of 800, 500, 300, and 1µm and are packed in capillaries with IDs of 800, 500, 300, and 150µm. There are columns with smaller IDs available; these are referred to as 'nanoflow' columns since separations are accomplished at low nanoliter per minute flow rates and have IDs of 100µm, 75µm, and 50µm.²⁰

 

Comparison between HPLC, RRLC, UPLC, UFLC and NANO LC^21–26

 

 

Application: -

1. Application HPLC:^27–30

The information that can be obtained using HPLC includes identification, quantification, and resolution of a compound. Preparative HPLC refers to the process of isolation and purification of compounds. This differs from analytical HPLC, where the focus is to Obtain information about the sample compound.

 

1. 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.

 

2. 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. The migration of the compounds and contaminants through the column need to differ enough so that the pure desired compound can be collected or extracted without incurring any other undesired compound.

 

3. Identification:

Generally, assay of compounds is carried using HPLC. The parameters of this assay should be such that a clean peak of the known sample is observed from the chromatograph. 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.

 

4. Other applications of HPLC:

Other applications of HPLC includes

Pharmaceutical applications

·       Tablet dissolution study of pharmaceutical dosages form.

·       Shelf-life determinations of parmaceutical products

·       Identification of active ingredients of dosage forms

·       Pharmaceutical quality control Environmental applications

·       Detection of phenolic compounds in Drinking Water

·       Identification of diphenhydramine in sedimented samples

·       Bio-monitoring of pollutant Forensics

·       Quantification of the drug in biological samples.

·       Identification of anabolic steroids in serum, urine, sweat, and hair

·       Forensic analysis of textile dyes.

·       Determination of cocaine and metabolites in blood Clinical

·       Quantification of ions in human urine Analysis of antibiotics in blood plasma.

·       Estimation of bilirubin and bilivirdin in blood plasma in case of hepatic disorders.

·       Detection of endogenous neuropeptides in extracellular fluids of brain.

 

Food and Flavor:

·       Ensuring the quality of soft drink and drinking water.

·       Analysis of beer.

·       Sugar analysis in fruit juices.

·       Analysis of polycyclic compounds in vegetables.

·       Trace analysis of military high explosives in agricultural crops.

 

Applications of RRLC:²⁸

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

·       It is applicable for Herbal produces examples: Panax and Epimedium species

·       It is Applicable for not only pharmaceuticals compound examples: Methanol, vitamins B6, B9, B12, Tri ethylamine but also for chemical compounds example: Acetanilide, Acetophenone, octanophenone, Tubufenozide

·       Scalability as a Function of Column Dimensions Using ZORBAX Rapid Resolution HT Columns for the Analysis of the Pharmaceutical Triamcinolone

·       Impurity Profiling with the Agilent 1200 Series LC System Part 1: Structure Elucidation of Impurities with LC/MS

·       Polycyclic Aromatic Hydrocarbon (PAH) Separations Using ZORBAX Eclipse PAH

·       The High-Resolution Reversed-Phase HPLC Separation of Licorice Root Extracts using Long Rapid resolution HT 1.8-um Columns

·       The Analysis of Benzodiazepines in Hair Using RRHT LC/MS/MS

·       Determination of Benzodiazepines in Oral Fluid Using LC/MS/MS More speed, better resolution and lower LOD using liquid chromatography and fluorescence detection - Comparing the 1100 Series LC to the 1200 Series Rapid Resolution system.[10]

 

Applications of UPLC:^31,32

1. UPLC in bioequivalence and bio analysis are:

·       In UPLC and LC instruments and software combine in a sophisticated and integrated system for bio analysis and bioequivalence studies, providing unprecedented performance and compliance support.

·       UPLC delivers excellent chromatographic resolution and sensitivity.

·       MS delivers simultaneous full-scan MS and multiple reaction monitoring. (MRM) MS data with high sensitivity to address matrix monitoring.

 

2. Drug Discovery:

UPLC improves the drug discovery process by means of high throughput screening, combinational chemistry, high throughput in vitro screening to determine physiochemical and drug’s pharmacokinetics.

 

3. High Throughput Quantitative Analysis:

UPLC coupled with time-of-flight mass spectroscopy give the metabolic stability assay.

 

4. Analysis of Dosage form:

It provides high speed, accuracy and reproducible results for isocratic and gradient analysis of drugs and their related substance. Thus, method development time decrease.

 

5. Analysis of Amino acids:

UPLC used from accurate, reliable and reproducible analysis of amino acids in the areas of protein characterizations, cell culture monitoring and the nutritional analysis of foods.

 

6. Determination of Pesticides:

UPLC couples with triple Quadra-pole tandem mass spectroscopy will help in identification of trace level of pesticides from water.

 

Applications of UFLC:³³

a)     Determination of iodiconazole in micro-dialysis samples

b)    Determination of podophyllotoxin in dermal and blood micro-dialysis samples of rats.

c)     Simultaneous analysis of fluoroquinolones and xanthenes derivatives in serum.

d)    Analysis of Isoflavones in Soy.

e)     Analysis of Catechins in Green Tea.

f)     Analysis of artificial colorants by UFLC mass spectroscopy.

g)    Separation of major components in PanaxGinseng.

 

Application of Nano LC:^34–37

a) Separation of Sulphonamides:

Nano-liquid chromatography coupled with mass spectrometry was used for the simultaneous determination of 18 sulphonamides by utilizing a capillary column (100 µm I.D.) stationary phase, used is Kinetex core-shell. A binary mobile phase, consisting of water and acetonitrile and both containing 0.1%(v/v) formic acid, was employed in a gradient mode at a flow rate (190nL/Min).

 

b) Separation of Peptides:

An integrated multidimensional nano- flow liquid chromatography platform with the combination of protein and peptide separation via online digestion by an immobilized enzymatic reactor was established, and successful applied for proteome analysis.

 

c)Discovery of Glycomics towards biomarker using Nano-LC:

Nano Flow LC, or nano-LC, significantly provides a highly sensitive and quantitative method of separating and profiling glycans.

 

d)Nano-LC for Glycobioanalysis:

Structural heterogeneity of glycoconjugates and glycans in biological matrices. C18, graphited carbon and amide-based stationary phases were adapted to nanoflow level and on chip format, leading to improved sensitivity of structural analysis and superior level of information on highly complex glycans and conjugate mixtures.             

 

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. In RRLC High Sensitivity - Low limit of detection, Excellent Reproducibility, Broad Applicability, Ease of Use - Easy setup. Columns with small internal diameters and / or short column lengths are more susceptible to extra-column band-broadening for high-speed separation in UPLC. Ultra-fast analysis means a significant enhancement in sample throughput (5- 10t times) and productivity compared to a conventional HPLC. Nano LC is the latest innovation in separation science in which detect ions can achieved at nano gram or lower levels.

 

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Received on 12.10.2022       Modified on 05.11.2022

Accepted on 28.12.2022   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2023; 13(1):53-58.