A REVIEW ON RAPID RESOLUTION LIQUID CHROMATOGRAPHY OF AGILENT1200 RRLC SERIES

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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Haripriya et al. SJIF Impact Factor 5.210 Volume 4, Issue 07, 1581-1595.

1 WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Haripriya et al. SJIF Impact Factor Volume 4, Issue 07, Review Article ISSN A REVIEW ON RAPID RESOLUTION LIQUID CHROMATOGRAPHY OF AGILENT1200 RRLC SERIES P.Haripriya*, Sharadha Srikanth, A.Ajitha and V.Umamaheswararao Department of Pharmaceutical Analysis and Quality Assurance, CMR College of Pharmacy, Kandlakoya (v), Medchal Road, Hyderabad , T.S, India. Article Received on 16 May 2015, Revised on 07 June 2015, Accepted on 29 June 2015 *Correspondence for Author P.Haripriya Department of Pharmaceutical Analysis and Quality Assurance, CMR College of Pharmacy, Kandlakoya (v), Medchal Road, Hyderabad , T.S, India. ABSTRACT In this Application Note an Agilent 1200 Series Rapid Resolution LC system was provide a liquid chromatography offering ultra fast and highest analysis speed high improved sensitivity. High temperature up to 100 C on certain columns allows more selectivity flexibility and reduces solvent viscosity to allow even faster separation. This is achieved by using sub-2 micron (STM) column particle which means that for high flow rates or long columns additional pressure is required to drive the mobile phase through the column. Detectors with high data rates preserve the resolution of Very fast peaks eluting from the RRLC The needed analytical instrumentation requires optimal cost-ofownership instruments with high reliability, high flexibility, and ease of use. column particle, Ultra fast. KEYWORDS: Rapid Resolution Liquid Chromatography, Small INTRODUCTION TO LIQUID CHROMATOGRAPHY Chromatography is a powerful analytical method that is widely used for the separation, identification and determination of the chemical components in complex mixtures. It was first demonstrated by the Russian botanist Milkhail Tswett in 1906 when he separated different plant pigments by passing an extract Of the leaves through a glass column packed with calcium carbonate the term chromatography came from Greek words, chroma meaning "color and graphein meaning to write". Tswett s invention went virtually unnoticed for several decades. Chromatography may be defined as the separation of a mixture of components based on the distribution coefficients for each component between two phases; a Vol 4, Issue 07,

2 stationary phase and a mobile phase. The mobile phase is liquid consisting of a solvent or mixture of solvents and the stationary phase is a solid support. [7] CLASSIFICATION OF CHROMATOGRAPHIC SEPARATIONS (1)Adsorption chromatography; this is the traditional separation Mode in which the stationary phase is solid and the mobile phase is liquid. The mixture components adsorb on the surface of the solid phase. The more strongly a solute is adsorbed, the slower it travels the column. (2) Partition chromatography; the stationary phase is liquid bonded to a solid Support and the mobile phase is a liquid. The separation of the mixture components is based on their equilibration between the liquid on the stationary phase surface and the mobile phase. In the liquid-liquid partition chromatography, if a polar stationary phase with a non-polar mobile is used, polar compounds are retained and separated. This is called normal- phase chromatography. If non-polar used, non-polar compounds are retained and separated. This is called reversed-phase chromatography. (3)In ion exchange chromatograph, the stationary phase consists of either cationic exchange or anionic exchange groups attached to polymeric or silica materials and The mobile phase consists of an electrolyte. (4) In size exclusion chromatography, the separation of a mixture of components is based on the size of the molecules. Larger molecules are excluded from the pores of the stationary phase particles and eluted first, where as small molecules are retained in the pores for a while be for elution. [7] TYPES OF HPLC CHROMATOGRAPHY TECHNIQUES 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) FEATURES OF RAPID RESOLUTION LIQUID CHROMATOGRAPHY (RRLC) The design concept of the 1200 Rapid Resolution LC (RRLC) system was to provide a liquid chromatography offering ultra fast and highest analysis speed and high improved sensitivity Vol 4, Issue 07,

and pressure at a minimum yet which retain full functionality for standard HPLC applications. It is the fastest and most efficient and flexible LC system in the world.

3 and pressure at a minimum yet which retain full functionality for standard HPLC applications. It is the fastest and most efficient and flexible LC system in the world. It has become an increasingly useful approach to achieve higher throughput, improved sensitivity and reduce costs. The RRLC system enable faster analysis ( theoretically up to 20x) than with conventional HPLC. This is achieved by using sub-2 micron (STM) column particle which means that for high flow rates or long columns additional pressure is required to drive the mobile phase through the column. The RRLC flow path is optimized to produce minimal backpressure and ZORBAX RRHT columns have an engineered particle size distribution that produces significantly less back pressure those other STM columns. High temperature up to 100 C on certain columns allows more selectivity flexibility and reduces solvent viscosity to allow even faster separation. High flow rates up to 5ml/min can be used for ultra -fast separations. The adjustable delay volume filly supports 2.1 to 4.6 mm i.d. columns. A low dispersion tubing kit and low Volume flow cells minimize peak dispersion for narrow bore Columns. Detectors with high data rates preserve the resolution of Very fast peaks eluting from the RRLC. [10] RRLC has been applied for the analysis of nutritional components (catechins) in green tea using the same approach. In this study, the following goals were achieved. 1) Demonstration of ease of conversion of a traditional HPLC method to a RRLC method and 2) To investigate the potential of RRLC and RRLC/MS on 1.8 µ m porous particles packed into short columns operating at a high flow rate compared to the performance of 5 µ m porous particles packed conventional columns. [10] Fig 1: Rapid resolution liquid chromatography (RRLP) Vol 4, Issue 07,

4 Fig 2: Schematic diagram of RRLC 2. 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) H=A+B/u + Cu A-Eddy s diffusion B- Longitudinal diffusion C-Concentration u- Linear Velocity Column efficiency: N=16RT²/W² N-number of theoretical plates RT-retention time W-path widt Resolution is proportional to the square root of N. But since N is inversely proportional to particle size ( Resolution (Rs) equation. Rs= 4 (α-1/α) (k/k+1) Equation.2 Resolution is proportional to the square root of N. But since N is inversely proportional to particle size (dp). N 1/dp Equation.3. Vol 4, Issue 07,

5 Efficiency (N) is increased by smaller Particle size (1.7µm). N is also inversely proportional to the square of the peak width(w).so as the particle size decreases to increase N and subsequently Rs, an increase in sensitivity is obtained. Also, peak height(h) is inversely proportional to the peak width(w). H 1/W Equation.4 An increase in sensitivity is obtained, since narrower peaks are taller peaks. Narrower peaks also mean more peak capacity per unit time in gradient separations, desirable for many applications. eg. peptidemapping. N L/dp Equation.5 Efficiency(N) is proportional to column length(l) and inversely proportional to the particle size.(14). Fig 3: van Demeter plot 3. INSTRUMENTATION RRLC analyses were performed on an Agilent 1200 SL HPLC equipped with a binary pump and a micro-vacuum degasser, a multi-wavelength (MW) detector, an auto-sampler, a thermostatted column compartment, and a Luna C 18-HST column (2.5_m,100mm 3.0 mm) from phenomenex (Torrance,CA,US). [11] DESCRIPTON To achieve lowest baseline noise, the standard delay volume configuration is Recommended for the 1200 Series RRLC pump module. Vol 4, Issue 07,

6 The injection volume and the sample dissolution solvent are important. Care must be taken that the compounds are focused at the top of the column, to avoid peak dispersion due to the injection, which would cause a reduced peak height. In order to achieve this, the sample should be dissolved in a solvent composition of lower elution strength than the mobile phase. The column temperature should not be too low, to avoid long retention of the peaks on the column. This also creates peak dispersion and a lower peak Height. Selection of the optimum detector cell depends on the id of the column being used. Typically, the longer the path length, the better the signal-to-noise ratio. The data rate of the UV detector should be selected according to the actual peak width. Higher than necessary data rates should be avoided because of higher noise levels. Available UV detectors are the Agilent 1200 Series Diode Array Detector SL Plus and the new Agilent 1200 Series Variable Wavelength Detector SL, with data rates of 160 Hz and significantly lower noise and drift levels. For highest sensitivity in the UV range, the new Agilent 1200 Series VWD SL Plus is the optimum choice [11] Fig: 4 Instrumentation of RRLC DEGASSER µ-degasser: Maximum baseline stability and minimum noise by continues vacuum source Vacuum Degasser: Flow rate: up to 10ml/min, Internal volume: 12 ml. Micro Degasser: Flow rate: up to 5ml/min, Internal volume: 1ml. [1] Vol 4, Issue 07,

Fig 5: Vacuum degasser Micro degasser WORKING OF AUTOSAMPLER IN RRLC During the injection routine of the autosampler, the sample loop, the inside of the needle, the seat capillary and the main

7 Fig 5: Vacuum degasser Micro degasser WORKING OF AUTOSAMPLER IN RRLC During the injection routine of the autosampler, the sample loop, the inside of the needle, the seat capillary and the main channel of the injection valve are in the flow path, and remain there throughout the duration of run. This means. These parts are flushed continuously with mobile phase during the complete analysis. It is only during aspiration of the sample that the injection valve is switched out of the flow path. In this position, the pump effluent is led directly to the column. Prior to injection, the outside surfaces of the needle are washed with fresh solvent. This is achieved using the flush port of the autosampler, and prevents contamination of the needle seat. The flush port of the autosampler is refilled with fresh solvent by a peristaltic pump that is installed in the autosampler housing. The flush port has a volume of about 680 μl, and the pump delivers 6 ml/min. Setting the wash time to 10 seconds means that the flush port volume is refilled more than once with fresh solvent, which is sufficient in most cases to clean the outside of the needle. Flushing and leaning of the autosampler is thus achieved so as to have no (or) zero carry over. [11] Fig6:Autosampler SL injection system Vol 4, Issue 07,

PUMPS A steady pump pressure (usually about 1000 2000 psi) is needed to ensure reproducibility and accuracy Proper maintenance of the pump system will minimize down time.

8 PUMPS A steady pump pressure (usually about psi) is needed to ensure reproducibility and accuracy Proper maintenance of the pump system will minimize down time. Commonly used pumps in RRLC are binary pumps and quaternary pumps. BINARY PUMP SL The pump has the facility for mixing two solvents for binary gradients and of course may be used in the isocratic mode. By choosing a system from the fast quaternary/binary LC system through to the ultra-fast rapid resolution system you can achieve new levels of productivity, processing more sample in less time, while maintaining and even improving data quality. Short columns with sub-2 µm particles offer a unique opportunity to dramatically reduce analysis time by increasing the flow rate without losing separation performance. In order to take full advantage of their power, you need a system that can do more than handle high back pressure. The answer is a fully integrated solution, optimized for system volumes, cycle times, detection rates and temperature control. Configurable delay volume (120µl and µl) for uncompromised support of narrow and std bore columns ( mm ID). Standard delay volume ( µl) Low delay volume (120 µl) ml/min flow rate range for highest speed on narrow and standard bore RRHT columns (full P range). 600 bar pressure for higher speed and resolution in RRLC. Electronic damping control for lowest baseline noise Perfect choice for fast and precise gradients using LC/MS, as well as UV-only systems Fully exploits the speed and separation potential of ZORBAX Rapid Resolution HT Columns. Fig 7: Binary Pump SL Vol 4, Issue 07,

Binary pump Flow range: 0.001 5 ml/min, for fast gradient analysis (column id: 2.1-4.

9 Binary pump Flow range: ml/min, for fast gradient analysis (column id: ) BENEFITS OF COLUMNS PACKED WITH SUB-2MICROPARTICLES FASTER CHROMATOGRAPHY There are several advantages of having short run times. High Throughput labs now have higher capacity and can analyze more samples in less time, also means lower costs. [1] HIGHER RESOLUTION Long columns packed with smaller particles result in higher efficiency and higher resolution. This is important for analysis of complex samples from metabolomics or proteomics. Also, applications such as impurity profiling can benefit from higher separation powder. Even the LC/MS analysis of drug in biological fluids can benefit from the higher peak capacity, because of the reduced interference from ion suppression. In general, higher separation power provides more confidence in the analytical results. [1] Fig 8: Peak capities of more than 700 can be achieved using a ZORBAX RRHT SB-C 18column (2.1 x 150 mm, 1.8 µm) to analyze tryptic digest of Bs. FRICTIONAL HEATING Forcing mobile phase through the column at higher pressure and higher flow rates generates heat. The resulting temperature gradients have an impact on the column efficiency. POWER= F.p F=Flow rate P = pressure Powerful column Thermostatting (for example, using a water bath) generates a strong radial temperature gradient, which leads to significant loss in column efficiency. Still-air-column Vol 4, Issue 07,

thermostatting reduces the radial temperature gradient and therefore reduces the efficiency losses, but a higher column outlet temperature has to be accepted.

10 thermostatting reduces the radial temperature gradient and therefore reduces the efficiency losses, but a higher column outlet temperature has to be accepted. At lower back-pressure, performance losses due to frictional heat are minimized so that 4.6/3mm inner sub-2-micron columns still deliver superior efficiencies compared with the respective 2.1 mm inner diameter columns [1] COLUMN 1) THERMOSTATTED COLUMN COMPARTMENT SL The heart of the system is the column where separation occurs 100 C for higher speed and resolution in RRLC. New low volume heat exchanger for minimized peak broadening on 2.1mm ID columns. 600 bar 2Ps/10Pt micro valve for minimum cycle times by ACR (alternating column regeneration, which saves wash & regeneration time) PCC (post-column cooling) and improved peltier temperature control (< +/ C) for minimized UV noise under most demanding conditions (< 50µAU at high flow rates and temperatures) Fig 9: Thermostatted Column Compartment Sl 2) COLUMN DIAMETERS Column optimized for 2.1mm, 3 and 4.6 mm I.D are available in RRLC for improving its efficiency. Vol 4, Issue 07,

1mm Id column with highest Uv sensitivity OPTIMIZED CONFIGURATION FOR 3 AND 4.

0mm ID columns with highest UV sensitivity.

11 Fig 10: Low delay configuration for 2.1 mm inner diameter columns Fig 11: medium delay volume configuration for 2.1mm Id column with highest Uv sensitivity OPTIMIZED CONFIGURATION FOR 3 AND 4.6 MM I.D. COLUMNS: Fig 12: Standard delay volume configuration for 3 and 4.0mm ID columns with highest UV sensitivity. DETECTORS The detector is used to sense the presence of a compound passing through and to provide an electronic signal to a data-acquisition device. Vol 4, Issue 07,

12 DAD/MWD SL s: (DIODE-ARRAY DETECTOR/MULTIPLE WAVE LENGTH DETECTOR) Up to 100% resolution gain in UFLC by 80Hz data rate Highest sensitivity in UFLC with < 50uAU noise by Low noise cell design and electronics, ETC (Electronic Temperature Control),PCC (Post column cooling using TCC SL) Support of 2.1, 3.0 and 4.6mm ID columns by 3 flow cells Standard: 13 ul / 10 mm, Semi- Micro: 5 ul / 6 mm, Micro: 2 ul / 3 m The Agilent 1200 Series diode-array and multiple-wavelength detectors: Dual lamp design for highest sensitivity from 190 to 950 nm; Programmable slit for easy optimization of sensitivity, linearity and spectral resolution; Low-noise electronics and electronic temperature control for lowest detection limits, even under unstable ambient conditions A range of nine flow cells for highest application flexibility; and Easy upgrade to the 80 Hz sampling rate for high speed separations. Multiple wavelength detector SL for ultra-fast multi-electrical analysis. It has 8signals, 80 HZ data rate. Diode Array Detector SL for multi-electrical analysis. It have 5 signals, 20 HZ data rate [5] VWD SL (VARIABLE WAVE LENGTH DETECTOR SL) VWD SL for ultra-fast, programmable single ë analysis, 1 signal, 13 Hz data rate Maximum resolution in UFLC by 55Hz sampling rate. Three flow cells for support of narrow and std bore columns ( mm ID), Std: 14ul / 10mm, Semi-micro: 5ul / 6mm, Micro: 1ul / 5mm [5] FIG 13: Influence of the detector rate on resolution DAD SL and MWD SL Vol 4, Issue 07,

TYPES OF DETECTORS AVAILABLE IN AGILENT SERIES Different UV detector flow cells for use with 2.1, 3.0 and 4.6 mm inner diameter columns are available.

13 TYPES OF DETECTORS AVAILABLE IN AGILENT SERIES Different UV detector flow cells for use with 2.1, 3.0 and 4.6 mm inner diameter columns are available. Fast UV and MS detectors with data rates up to 160 Hz (1200 Series VWD SL Plus), 80HZ (1200 series DAD SL, MWD SL) and up to 40 HZ for MS application are available. The 1200 Series fluorescence detector (FLD) with 37 Hz data acquisition, and the 1200 series refractive index detector (RID) are also compatible with the Agilent 1200 series RRLC system. A stepwise upgrade from 1100 series to 1200 series RRLC is possible. [9] FIG NO: 14.Diagrammatic representation of differences/changes between HPLC and RRLC Advantages over HPLC The major high - throughput RRLC are the increase in throughput. The reduction in the analysis cost. The shortening in analysis time is due to the use of a shorter column length high resolution separation capability ultra fast and highest analysis speed It is most improve sensitivity High temperature up to 100 C on certain columns allows more selectivity flexibility It is offers true fast LC analysis [11] Vol 4, Issue 07,

14 6. Applications of RRLC 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 [9] 7. CONCLUSION In this study, 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. RRLC separations could be easily achieved from many traditional HPLC methods. Most applications of these small particle columns have been in the area of pharmaceutical analysis. Using a 1200 RRLC system equipped with higher acquisition rate of detector, low dead volume system configuration and combined with a high-pressure HPLC system with 600 bar pressure capability, the ultrafast RRLC analysis could be done with satisfactory analytical precision. In an isocratic example, one cycle analysis time by traditional HPLC/DAD method that required 25 min could be shortened to a RRLC/MS method to 0.8 min, a factor of 31. The same method transfer approach was carried out in a gradient separation. RRLC technique is useful for not only pharmaceuticals but also for chemical compounds currently analyzed by conventional HPLC on 250mm 4.6 mm i.d. Vol 4, Issue 07,

15 columns. In the future, the fast RRLC technique will be used more widely in other HPLC fields. 8. REFERENCES 1. Angelika Gratzfeld-Huesgen, Michael Frank, Christian Gotenfels. Agilent Series RRLC Optimization Guide., 1200; Chen F, Ying GG, Yang JF, Zhao JL, Wang L, J Environ Sci Health B., 2010 Oct; 45(7): Frank.M, ìachieving fastest analyses with Agilent 1200 Se-rise RRLC System and Application Note Publication Number 2006; EN, March 4. Gerber. F, Krummen. M, Potgeter.H, Roth A. Siffrin. C and Spoendli c.j chromatograph., 2004; A1036: Greibrokk T. and Andersen T., J. Chromatograph, 2003; A1000: Ian Acworth, Darwin Asa, Eddie Goodall1, John Christensen, and Ryan McCarthy Available from: URL: 9. Pickering Laboratories, Application Note, Publ. No B-CA 5, Tatsunari Yoshida, Ronald E. Majors and Hiroki Kumagai, Chromatography, 2007; Vol.28 (No.2). 11. Takakura Cho, Hachioji Shi Tokyo Agilent Technologies, Inc Japan. 12. Agilent Technologies, Inc 2850 Centerville Road Wilmington, Delaware USA. 13. URL: http//schimadzu.co.uk. 14. Van Demeter JJ, Zuiderweg EJ, Klinkenberg A.Longitudinal diffusion resistance to mass transfer as causes of non ideality in chromatography. Chem. Eng. Sci., 1956; 5: Yuan-Chun Maa,b,, Xiao-Qiang Wanga,b, FeiFei Houa, Jie Ma, Mai Luoa, Alice (Chena, Peter Jin, Shane Lua, Iris Xuaa (RRLC) analysis and studies on Journal of Pharmaceutical and Biomedical Analysis., 2011; 54 : Vol 4, Issue 07,

LC walk-up system using the Agilent 1200 Series LC Method Development Solution and Agilent Easy Access software. Application Note

LC walk-up system using the Agilent 12 Series LC Method Development Solution and Agilent Easy Access software Test of reaction kinetics, column scouting, and impurity checks with one LC system and up to