UPLCUFLCRRLCNANO 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 µm, there is a significant gain in efficiency.

HPLC Instrumentation and Techniques

Instrumentation

Mobile phase reservoirsConstruction pump material

PumpsRecriprocating

SyringeManual Injection

Automated Injection

Packings

Measure column performance

Column care and use

SolventDelivery

SampleIntroduction

Column Packingsand harware

Detectors

UV-Vis

Fluorescence

Refractive Index

ConductivityVolatametry/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 μm 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 UPLC™BEH 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 μm 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 charecteristics HPLC UPLC

particle size 3 to 5m less than 2m

Analytical column XTerra c18,Allitma c18

ACQUITY UPLC BEH C18,c8,RP SHEILD,phenyl

column Dimensions 150× 3.2 mm 150× 2.1mm

column temperature 30 c 65 c

Injection volume 5 µL 2µL

Flow Rate 3.0 mL/min 0.6mL/min

Needle wash methanol Strong Needle Wash: methanolWeak needle wash:ACN:H2o 10:90

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 2.2µm 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 Å

• 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• 2.5µm partcle size• Significantly lower backpressure compared to other 2µm

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 2.2µm 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 : 5µl 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 :2µl

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 RRLC

column ID(mm) 2.1- 4.6 2.1- 4.6

Particle size (µm) 3,5 1.8

Pressure (psi) 3000 9000

Flow rate (ml/min) 0.6-1.2 0.2- 2.0

Temperature (0c) upto 40 upto 100

Faster Analyses with RRLC Quaternary Amines by HPLC-CAD

System: Agilent 1200 RRLCColumn: Shiseido MG C18, 4.6 x 250 mm (5 μm)

Mobile Phases A: Water, 0.1% Formic acid B: AcetonitrileFlow 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 μL

Run Time: 25.00 minutes

Corona CAD: N2 Pressure: 35.0 psi

Filter: High

Nebulizer Temperature: 30 °C

APPLICATIONS OF RRLC

Quaternary Amines by RRLC-CADSystem: Agilent 1200 RRLCColumn: Waters UPLC BEH C18, 2.1 x 50 mm (1.7 μm)Mobile Phases A: Water, 0.1% Formic acid B: Acetonitrile

Flow Rate : 0.65 mL/minGradient: 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.), 30°C (post-col.)

Injection Volume: 2 μL

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 µL/min, second dimension 50-1000 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 ColumnPacked columns are made by “stuffing” the capillary with silica-modified particles of 3–5 μm. Though recently, particles of even smaller sizes 1.5–1.8 μm 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 ColumnMonoliths 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 ColumnIn 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

REFERENCESIJPQA 2010, JAN-MAR VOL.2,ISSUE 1(19-25)

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WWW.ESAINC.COM

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