UPLC UFLC RRLC NANO LC

UNDER THE GUIDANCE OF

Dr. R. GOVINDARAJAN

PRESENTED BY
V.RAJESH Y10MPH232

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 1/column efficiency).

H=A+B/u+Cu

Since particle size is one of the variables, a van Deemter curve can be used to investigate chromatographic performance. According to the van Deemter equation, as the particle size decreases to less than 2.5 m, there is a significant gain in efficiency.

HPLC Instrumentation and Techniques Mobile phase reservoirs Solvent Construction pump material Delivery Recriprocating Pumps Syringe Sample Introduction Manual Injection Automated Injection

Instrumentation

Packings Column Packings and harware Measure column performance Column care and use UV-Vis Detectors Fluorescence Refractive Index Conductivity Volatametry/Amperometry

ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC)

COLUMNS IN UPLC
 ACQUITY UPLC BEH C18 and C8 columns were designed to be the universal columns of choice for most UPLC separations by providing the widest pH range  ACQUITY UPLC BEH C18 and C8 columns incorporate trifunctional ligand bonding chemistries which produce superior low pH stability and ultra-low column bleed.  This low pH stability is combined with the high pH stability of the 1.7 m BEH particle to deliver the widest usable pH operating range.  These bonding chemistries and particle synthesis innovations produce the sharpest peaks, highest efficiencies.

ACQUITY UPLC BEH Shield RP18 Columns

 Embedded polar group (Shield) RP columns contain stationary phases that combine the hydrophobicity of an alkyl ligand with the hydrophilicity of an embedded polar group.  Embedded polar group columns include alternate selectivity to that of alkyl reversed-phase columns, excellent peak shape for bases and aqueous mobile phase compatibility.  The unique selectivity of Shield RP phases, especially for polyphenolic compounds, has been attributed to the embedded polar groups acting as hydrogen bond acceptors.  ACQUITY UPLCBEH Shield RP18 columns are designed to provide selectivities that complement the ACQUITY UPLC BEH C18 and C8 phases.

ACQUITY UPLC BEH Phenyl Columns

 ACQUITY UPLC BEH Phenyl columns were intelligently designed to overcome these weaknesses and provide complementary selectivities, pH stability and excellent peak shape for all compounds  ACQUITY UPLC BEH Phenyl columns utilize a trifunctional C6 alkyl tether between the phenyl ring and the silyl functionality.  This ligand, combined with the same proprietary endcapping processes as the ACQUITY UPLC BEH C18 and C8 columns, provides long column lifetimes and excellent peak shape.  This unique combination of ligand and endcap on the 1.7 m BEH particle creates a new dimension in selectivity and efficiency for challenging UPLC separations.  ACQUITY UPLC BEH columns provide column lifetimes under UPLC conditions which meet and/or exceed HPLC column lifetimes run under HPLC

COMPARISON OF HPLC AND UPLC HPLC UPLC charecteristics

particle size Analytical column 3 to 5m XTerra c18,Allitma c18 150 3.2 mm 30 c 5 L 3.0 mL/min methanol less than 2m ACQUITY UPLC BEH C18,c8,RP SHEILD,phenyl 150 2.1mm 65 c 2L 0.6mL/min Strong Needle Wash: methanol Weak needle wash:ACN:H2o 10:90

column Dimensions column temperature Injection volume Flow Rate Needle wash

APPLICATIONS OF UPLC
Analysis of natural products and traditional herbal medicines Identification of metabolite Bioanalysis/bioequivalence studies Impurity profiling USE OF UPLC SYSTEM
1.Elevated-temperature chromatography also allows for high flow rates by lowering the viscosity of the mobile phase, which significantly reduces the column back pressure. 2.Monolithic columns contain a polymerized porous support structure that provide lower flow resistance than conventional particle-packed columns

ULTRA FAST LIQUID CHROMATOGRAPHY

Ten times higher speed and three times better separation. Uflc offers outstanding speed and separation even at normal pressure levels, objectives that were difficult for conventional systems. By maximizing the column and performance of the entire system,uflc minimizes the deviation from the van deemter theory

UFLC COLUMNS
Uflc silica column Available as 1.8 and 2.2m particle sizes Available in analytical and preparative HPLC scale column dimensions and particle sizes Wide pore size selection - 60, 120, 200, 300, 500, 1000 & 2000 HP Silica phase is made of activated hydroxyl (-OH) functional group. Used for both normal phase and HILIC applications e.g. LC/MS analysis of nitrogen-containing compounds. C18 uflc column C18 reverse-phase UFLC/UHPLC Column 2.5m partcle size Significantly lower backpressure compared to other 2m columns Columns to support easy scale-up of UFLC separations to preparative scale

 Cyano column Available as 1.8 and 2.2um particle sizes Available in analytical and preparative HPLC scale column dimensions and particle sizes Monomeric and fully endcapped phase structure; % carbon load - 7% Propyl cyano functional group pH stability 2 - 8.5 amino column Available as 1.8 and 2.2m particle sizes Available in analytical and preparative HPLC scale column dimensions and particle sizes Monomeric and fully endcapped phase structure; % carbon load - 4% Propyl amino functional group pH stability 2 - 8.5

APPLICATIONS OF UFLC
Analysis of isoflavones in soy column: shimpack XR-ODS mobile phase : A. 0.1% formic acid aqueous solution B. 0.1% formic acetonitrile solution flow rate : 1.2ml per min column temperature : 400 c injection volume : 5l column pressure : 28.8 mpa

Analysis of catechins in green tea

Column : shimpack XR- ODS Mobile phase : A. 0.1%formic acid aqueous solution/THF = 95/5 B.Acetonitrile Flow rate :0.5ml/min Column temperature : 50o c Injection volume :2l column pressure : 19mpa

RAPID RESOLUTION LIQUID CHROMATOGRAPHY

Typically RRLC (Rapid Resolution Liquid Chromatography )columns have lower particle size - 1.8 microns compared with 2 to 10 micron conventional columns. Because of their lower particle sizes they operate at higher pressure (600 Bar ) levels than normal columns (400 Bar).

 Rapid Resolution Liquid Chromatography (RRLC) has become an increasingly useful approach to achieve higher throughput, improve sensitivity and reduce costs. Rapid Resolution LC system enables faster analysis (theoretically up to 20x) than with conventional HPLC while maintaining equivalent resolution. This is achieved by using sub-2 micron column particle chemistry and high flow rates. Often higher temperatures are employed to minimize system back-pressure.

 High resolution chromatography 90,000 plates in 4 minutes Ultra-fast separations up to 20 times faster Full compatibility with existing HPLC methods More detection capabilities from UV-visible and ELSD through LC/MS Near-zero sample carryover for uncompromised data quality Highest system flexibility for automated method development

COLUMNS IN RRLC
Thermostatted Column Compartment SL Temperature range: 10 C below ambient to 100 C Two independent heat exchangers allow pre-column heating and post-column cooling for lowest detection limits 400. PARAMETERS IN HPLC AND RRLC
HPLC column ID(mm) Particle size (m) Pressure (psi) Flow rate (ml/min) Temperature (0c) 2.1- 4.6 3,5 3000 0.6-1.2 upto 40 RRLC 2.1- 4.6 1.8 9000 0.2- 2.0 upto 100

APPLICATIONS OF RRLC
Faster Analyses with RRLC Quaternary Amines by HPLC-CAD
System: Agilent 1200 RRLC Column: Shiseido MG C18, 4.6 x 250 mm (5 m) Mobile Phases A: Water, 0.1% Formic acid B: Acetonitrile Flow Rate : 1.00 mL/min Gradient : T= 0 min 10% B, T= 15 min 90% B, T= 20 min 90% B, T= 22 min 10% B. Column Temperature: 40 C Injection Volume: 2 L Run Time: 25.00 minutes Corona CAD: N2 Pressure: 35.0 psi Filter: High Nebulizer Temperature: 30 C

Quaternary Amines by RRLC-CAD

System: Agilent 1200 RRLC Column: Waters UPLC BEH C18, 2.1 x 50 mm (1.7 m) Mobile Phases A: Water, 0.1% Formic acid B: Acetonitrile Flow Rate : 0.65 mL/min Gradient: T= 0 min 10% B, T= 3 min 90% B, T= 4 min 90% B, T= 4.5 min 10% B Column Temperature: 50 C (pre-col.), 30C (post-col.) Injection Volume: 2 L Run Time: 6.00 minutes Corona ultra2 Pressure: 35.0 psi: N Filter: High Nebulizer Temperature: 30 C

NANO LIQUID CHROMATOGRAPHY

Optimized for ultra-low flow rates of first dimension 1-20 L/min, second dimension 501000 nL/min. Novel pumping system eliminates the need for flow splitting, providing excellent run-to-run reproducibility. Use automated peak parking for extended MS/MS analysis to improve peptide sequence coverage. Take advantage of nanospray sensitivity. Active flow control of each mobile phase ensures precise gradient delivery. Quickly responds to changes in flow rate set point in less than two seconds. Reduce solvent consumption by 99%!

COLUMNS IN NANO LC
Three basic types of capillary columns used in nano-liquid chromatography: packed, monolithic, and open tubular.

Packed Capillary Column

Packed columns are made by stuffing the capillary with silicamodified particles of 35 m. Though recently, particles of even smaller sizes 1.51.8 m were successfully employed in ultra performance LC (UPLC). Such a small particle size provides nano-liquid chromatography systems with higher efficiency, resolution, selectivity, and shorter analysis time; however, it does increase the backpressure. So far, the application of packed capillary columns is the most explored option in nano-liquid chromatography.

 Monolithic Capillary Column

Monoliths are a block of continuous materials made of highly porous rods with two types of pore structures (macropores and mesopores of different sizes), which allow the use of higher flow rates and thus reduces the analysis time.

Presently four types of monolithic capillary columns can be found: particle fixed, silica based, polymer based, and molecular imprinted monolith. Up-to-date there is not much research information on application of monolithic capillary in nano-LC.

 Open Tubular (OT) Capillary Column In open tubular liquid chromatography column, the capillary wall is coated with highly permeable porous material that serves as the stationary phase. The OT capillary has lower sample loading capacity of the column, because only a small surface area is available for analyte interaction that can result in column overloading causing peak asymmetry and poor efficiency.

APPLICATIONS OF NANO LC
Protein identification and functional analysis require new analytical tools and techniques that offer high throughput and enhanced detection sensitivity for identification of low-abundance proteins.

ADVANTAGES
1.Significantly reduces solvent consumption and subsequent waste production. 2.ID diameter reduction increases sensitivity and/or less sample requirement. 3.Decrease in column bead size (packing) narrowers the peak width of chromatogram due to better separation efficiency. 4.Does not increase system pressure

REFERENCES
IJPQA 2010, JAN-MAR VOL.2,ISSUE 1(19-25) WWW.SHIMADZU.NET WWW.ESAINC.COM WWW.EKSIGENT.COM WWW.AGILENTED.COM

THANK U