hplc separation fundamentals120109 - agilent separation fundamentals bill champion application...

HPLC Separation pFundamentals

Bill ChampionApplication Engineer

Agilent Technologies, IncDecember 1, 2009

Separation fundamentalsAgilent Restricted

12/1/2009

Presentation Outline

Major HPLC modes

Key Equations• Resolution

D t• van DeemterCommon terms & definitions

Key parameters & conditions that affect themKey parameters & conditions that affect them• efficiency, selectivity, and retentionRole of pressurep

• Sub-2um

Separation fundamentals Agilent Restricted

12/1/2009

Presentation Objectives

Understand physical significance of chromatographic parameters especiallychromatographic parameters, especially Retention Factor and Resolution

Understand effect of role of Selectivity andUnderstand effect of role of Selectivity and Column Efficiency in improving Resolution

Column Selection

Parameters that affect column pressure


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Separation Techniques

Hydro -philic

VolatileC b li

Amino acids Inorganic ions

SugarsSyntheticCarboxylicacids

Suga sSugarsalcohols

food dyesGlyphosate

Sulfonamides

AldehydesKetones

GlycolsEnzymes

Aflatoxins

A ibi i

PG, OG, DGphenols

Fatty acids

Polarity

AntibioticsNitriles

Nitrosamine

Fatty acids

FlavonoidsBHT, BHA, THBQantioxidents

Alcohol PAHs Anabolica Natural food dyesOrgano-phosphoruspesticides Aromatic amines F t l bl it iTMS p Aromatic amines Fat soluble vitamins

PCBsTMSderivativesof sugar

Essential Oils Polymer monomers Triglycerides Phospholipids

A ti t

Epoxides

F tt id

Volatile – Gas Phase Nonvolatile - Liquid Phase

Hydro-phobic

C2-C6 hydrocarbons Aromatic estersFatty acidmethylesters


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Volatile Volatility Nonvolatile

Major Separation Modes of HPLC A ReviewA Review

There are four major separation modes that are d t t t dused to separate most compounds:

Reversed-phase chromatography (most popular)Normal-phase and adsorption chromatographyIon exchange chromatographySize exclusion chromatographySize exclusion chromatography

….Let’s look briefly at each mode….Let s look briefly at each mode


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Normal Phase or Adsorption ChromatographyK P i tKey Points

Column packing is polar• silica (strongest)>amino>diol>cyano (weakest)

Mobile phase is non polarMobile phase is non-polar • hexane, iso-octane, methylene chloride, ethyl acetate, etc.

Polar compounds more retainedRetention decreases as polarity of mobile phase increases

Reasons to choose normal phaseResolve strongly retained hydrophobic samples.Isomer separation.Sample injection solvent is non-polar.Recovery in non-polar solvents is desirable.

Separation of Nitroanilines on HPLC Column packed with silica gel using hexane (mobile phase component A) mixed with methylene chloride (mobile phase component B)


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Ion Exchange Chromatography

In ion exchange:Column packing contains ionic groups,

( lf t t t lk l i )(e.g. sulfonate, tetraalkylammonium)Mobile phase is an aqueous buffer (e.g. phosphate, formate, etc.)Used infrequentlyq ySimilarities to ion-pair chromatographyWell suited to the separation of inorganic and organic anions and cations in aqueous solution

Basic proteins on strong cation exchanger (-SO3

-)1 RNA polymerase

…cations in aqueous solution.

1. RNA polymerase2. Chymotrypsinogen3. Lysozyme


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Size Exclusion Chromatography (SEC)There are two modes: • non-aqueous SEC [sometimes termed Gel Permeation Chromatography (GPC)]• aqueous SEC [sometimes referred to as Gel Filtration Chromatography (GFC)]N i t ti b t th l d d ki t i lNo interaction between the sample compounds and packing material• Molecules diffuse into pores of a porous medium• Molecules are separated depending on their size relative to the pore size

molecules larger than the pore opening do not diffuse into the particles while molecules smallermolecules larger than the pore opening do not diffuse into the particles while molecules smaller than the pore opening enter the particle and are separatedlarge molecule elute first, smaller molecules elute later

The mobile phase is chosen mainly to dissolve the analyteU d i l f l h t i ti d f t i

Gel Permeation Chromatogram of Polybutadiene polymer on non-aqueous SEC (GPC) column;

Used mainly for polymer characterization and for proteins.

aqueous SEC (GPC) column; The monomers elute after the polymer.Column: PLgel mixed-D gel Mobile phase: Tetrahydrofuran (THF)


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Mechanism of SECMolecules (A,B) Enter Pores( )

A

B Partial entryFull entry

CPacking Pore

Molecule (C) Will be Excluded from Pores

C

Molecules must freely enter and exit

Sign

al C B Ay

pores to be separated. Largest molecules elute first, followed by intermediate size molecules and finally the smallest molecules elute last.


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Time

Reversed-Phase Chromatography (RPC)Principle: Partition of analytes between polar mobile phase and non-polar stationary phase

Nonpolar (nonspecific) interactions of analyte with hydrophobic (or lipophilic)Nonpolar (nonspecific) interactions of analyte with hydrophobic (or lipophilic) stationary phase:

– C18, C8, Phenyl, C3, etc.

Different sorption affinities between analytes results in their separationDifferent sorption affinities between analytes results in their separation– More polar analytes are less retained – Analytes with larger hydrophobic part are retained longer

Mobile phase: water (buffer) + water-miscible organic solventMobile phase: water (buffer) water miscible organic solvente.g. MeOH, ACNCan be used for non-polar, polar, ionizable and ionic molecules

G di t l ti i ft dGradient elution is often used


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Chromatography Terms are All Around UsBut what do they meanBut what do they mean…..

Gradient Steepness

Peak Capacity

RRLC

UPLC


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Some Basic Chromatography Parameters

• Retention Factor (k) Capacity Factor• Retention Factor (k), Capacity Factor (k’)

• Selectivity or Separation Factor (α)

• Column Efficiency as• Column Efficiency as Theoretical Plates (N)

( )• Resolution (Rs)

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Definition of ResolutionDefinition of Resolution

Rs =tR-2 - tR-1 =

∆tRRs (w2 + w1)/2 w

Resolution is a measure of the ability to separateResolution is a measure of the ability to separate two components

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Chromatographic TermsResolution

The distance between two neighboring peaksR = 1.5 is baseline resolutionR = 2 is highly desirable during method developmentR = 2 is highly desirable during method development

R = 2 (tR2 – tR1)( + )

Resolution is reduced based on how fat or id th k thi i b tt !(w1 + w2) wide the peaks are, so thin is better!

t00

Original> N value

> k’ values< k’ values >α value


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Resolution … Determined by 3 Key Parameters –y y

Efficiency, Selectivity and Retention

The Fundamental Resolution Equation

N = Column Efficiency – Column length and particle size

α = Selectivity – Mobile phase and stationary phase

k = Retention Factor – Mobile phase


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Chromatographic ProfileEquations Describing Factors Controlling REquations Describing Factors Controlling RS

Retention Factor

k =(tR-t0)

t0

α = k2/k1

Selectivity

α k2/k1

Theoretical Plates-Efficiency

N = 16(tR / tW)2

= 5.54(tR / W1/2)2


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Retention Factor (k), Capacity Factor (k’)

Separation is an Equilibrium Process

Sample Partitions between Stationary Phase and MobilePhase

K = C /CK = Cs/CmCompound moves through the column only while inmobile phase.p

Separation occurs in Column Volumes. (Flow is volume/time)(Flow is volume/time)

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Retention Factor (k), Capacity Factor (k’)

k =t0

tR – t0K = Cs/Cm =>=>

k is measure of number of column volumes required to l t d

0

elute compound.

Fundamental, dimensionless parameter that describesth t tithe retention.

k = 1 to 20 - OK; k = 3 to 10 - Better; k = 5 to 7 - Ideal

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Chromatographic Terms Retention FactorRetention Factor

Two compounds can be separated if they have sufficiently different retention factors – k values

k =t0

tR – t0

tR2tR3tR1t00


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Chromatographic Terms Selectivity factor

Alpha, α (separation factor, relative retention, ratio of capacity factors) this is used to measure how far apart the k’ values offactors) – this is used to measure how far apart the k’ values of two peaks are and if the separation can be achieved

α =k’2α =k’1

k’2 > k’1 α > 1 for a separation to take place

Big ⟨


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More Chromatographic Terms Efficiencyy

N - Number of theoretical plates – This is one case where more is better! “Plates” is a term inherited from distillation theory. For LC, it is a measure of the relative peak broadening for an analyteit is a measure of the relative peak broadening for an analyte during a separation – w (from our chromatogram).

Column length

N = 16tRw

2N =

LHor

Column length

HETPHETP

A Single Real Plate

A Number of Theoretical Plates


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A Single Real Plate

Resolution as a Function of Selectivity, C ffColumn Efficiency, or Retention

7.00

Selectivity Impacts Resolution Most

5.00

6.00 • Change bonded phase • Change mobile phase

α

3.00

4.00

Res

olut

ion Increase N

Increase Alpha

Increase k'Rs = N½/4 • (α-1)/α • k’/(k’+1) N1.00

2.00

Rs N /4 (α 1)/α k /(k 1) Nk

0.001 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Plates: 5000 10000 15000 2000025000Alpha: 1.10 1.35 1.60 1.85 2.1

k’: 2.0 4.5 7.0 9.5 12.0

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Start Method Development with RRHT Columns:Different ZORBAX C18 Bonded Phases for Max Selectivity

Eclipse Plus C18

M bil h (69 31) ACN t

1 23

41st choiceBest Resolution& Peak Shape

StableBond SB C18

Mobile phase: (69:31) ACN: waterFlow 1.5 mL/min.Temp: 30 °CDetector: Single Quad ESI positive mode scan Columns: RRHT

1 2 3 4 5

1 234

2nd choiceGood alternate selectivity due to non-endcapped

Eclipse

SB-C18 Columns: RRHT 4.6 x 50 mm 1.8 um

Sample:1. anandamide (AEA)2 P l it l th l id (PEA)1 4

min1 2 3 4 5

pp

3rd choice Eclipse XDB-C18

2. Palmitoylethanolamide (PEA)3. 2-arachinoylglycerol (2-AG)4. Oleoylethanolamide (OEA)

1 23

4

1 2 3 4 5

3 choiceGood efficiency & peak shapeResolution could be achieved

4th choice Multiple bonded h f t

Extend-C181 2,3 4

4 choiceResolution not likely,Other choices better, for this separation.

phases for most effective method development.Match to one you’re


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1 2 3 4 5currently using.

Small Differences in α Can Provide Large Differences in ResolutionDifferences in Resolution

Eclipse Plus C8Greatest Resolution for this sample

caffeine

barbital

sulfametho

Tailing factors:Peak 1: 1.03Peak 2: 1 01

E li Pl C18

α Resolutionpeaks 1,2 1.68 6.12peaks 2,3 1.4 4.97

e oxazole

Peak 2: 1.01Peak 3: 1.00

min0 1 2 3 4

Eclipse Plus C18

α Resolutionpeaks 1,2 1.33 3.55

k 2 3 1 31 3 82

Tailing factors:Peak 1: 1.07Peak 2: 1.03Peak 3: 1.04

Eclipse XDB-C8

peaks 2,3 1.31 3.82

Tailing factors:P k 1 1 07

min0 1 2 3 4

α Resolutionpeaks 1,2 1.43 4.15peaks 2,3 1.29 3.55Cols: 4.6 x 150, 5 um

m.p.: 40% MeOH, 60% water

Peak 1: 1.07Peak 2: 0.96Peak 3: 1.01


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min0 1 2 3 4

If α Has the Most Impact, Why Focus on N?It’s EasyIt s Easy

High plate number (N) provides:• Sharp and narrow peaks

• Better detection

• Peak capacity to resolve complex samples

But…• Resolution increases only with the square root of the plate number. • Plate number increase is limited by experimental conditions (analysis time, ..pressure)

Selectivity (α) helps best but Is difficult to predict so methodSelectivity (α) helps best but… Is difficult to predict, so method development is slower (experience helps, model retention)

Note: Software supported, optimization for separation of multi-component mixtures can d th d d l t ti (Ch S d D L b)


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reduce method development time (ChromSword, DryLab)

Peak Width Decreases with Increasing N –C l Effi iColumn Efficiency

N = 5000 theoretical plates 5um

N = 10,000 theoretical plates 3.5um

N = 20,000 theoretical plates 1 8um

Time (min)1 3 5 7 9 11 13 15

1.8um


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Peak Capacity Better Peak Capacity – More Peaks Resolved

Peak capacity is the number of peaks that can be separated (at a specified resolution, example R=1) by a given system (think column length, particle size) in a given amount of time.

It is another measure of efficiency.4

55μm 2 peaks fit

1.8μm

Rs = +50%

3 peaks fit1/3 More!!


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Decreasing Particle Size5 μm

8 min, 170 bar

3 5 μm

6 min, 204 bar

3.5 μm

3.4 min, 300 bar1.8 μm

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Smaller Particle Size Columns Improve R l ti B t P IResolution – But Pressure Increases

Up to 60% higher resolution than in conventional HPLC

7 Impurities

All 7 Baseline Separated!

4 Impurities

2 Not Baseline Separated!

Customer Example Isocr. Impurity Method

Zoom of critical time

7 Impurities

6 Not Baseline Separated! Separated!Separated!

range @ 7minSeparated!

4.6 x 150, 1.8um490 b

4.6 x 150, 5um93 b

4.6 x 150, 3.5um165 b 490 bar

N = 28,669 Rs = 1.80 (+57%)

93 barN = 7,259Rs = 1.15

165 barN = 14,862Rs = 1.37


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Putting it Together The van Deemter Equationq

Hei

ght

H

Sum Curve: van-DeemterH = A + B/u + C uLarge Particle

SmallParticle

H = L/N

Plat

e H Sum Curve: van Deemter

Resistance to Mass TransferH

Longitudinal diffusion Eddy Diffusion

H min

Linear Velocity u

The smaller the plate height, the higher the plate number and the greater the

u opt


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p g , g p gchromatographic resolution

Van Deemter Curve : HETP vs. Volumetric Flow Rate

0.0030Column: ZORBAX Eclipse XDB-C18 Dimensions: 4.6 x 50/30mmEluent: 85:15 ACN:WaterFlow Rates: 0 05 5 0 mL/min

H = A + B/u + Cu

0.0020

0.0025Flow Rates: 0.05 – 5.0 mL/minTemp: 20°CSample: 1.0μL Octanophenone in Eluent

(cm

)

0.0010

0.0015

HET

P 5.0μm3.5μm1.8μm

0 0000

0.0005

0.00000.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Volumetric Flow Rate (mL/min)Smaller particle sizes yield flatter curves minima shift to higher flow rates


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(mL/min)Smaller particle sizes yield flatter curves, minima shift to higher flow rates

Columns Packed with Smaller Particles ffProvide Higher Efficiency

sub 2 micron

3 micron N 1/(dp)α

N

5 micron P 1/(dp)2α

10 micron

VelocityVelocity

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Key Chromatographic Parameters I t d b P ti l SiImpacted by Particle Size

OptimumParticle Diameter

Efficiency

Pressure Analysis Time

Peak Volume

Pressure Analysis Time


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What About Pressure? Pressure Increases Exponentially with Decreasing Particle Size

ΔP =η L v

Equation For Pressure Drop Across an HPLC Column

ΔP = Pressure Drop

ΔP =θ dp

2 Many parameters influence column pressure

Particle size and column l th t iti lΔP = Pressure Drop

L = Column Length

= Fluid Viscosityη

length are most criticalLong length and smaller

particle size mean more resolution and pressureL = Column Length

v = Flow Velocityd = Particle Diameterp

resolution and pressureWe can now handle the

pressure

= Dimensionless Structural Constant of Order 600 For Packed Beds in LC

θ

p


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Mobile Phase How Does it Impact Pressurep

1. Solvent viscosity – lower viscosity results in lower pressure

• Acetonitrile < Methanol The difference between MeOH and ACN can be dramatic and is the first thing to change if lower pressure is needed.

• Water < Buffer While buffers increase viscosity, the organic selected is more critical. Make sure the buffer is soluble in the organic at all points in the run (gradient).

2. % of organic solvent – there is a pressure maximum and minimum for organic:aqueous mobile phases and it differs depending on the organic

• A 2.1 x 100mm column can be used with ACN below 400 bar, especially with slightly elevated temperature.

• But with MeOH you will need the 600 bar RRLC systems for almost all MeOH water mobile phases.


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Calculated Effect of Particle Size on K Ch t hi P tKey Chromatographic Parameters

dp Flow L Vm P P tR t0pμm mL/min cm mL psi bar Min Min

2 1.0 6 0.6 2500 172 3.6 0.6

3 1.0 10 1.0 1850 128 6.0 1.0

5 1.0 15 1.5 996 69 9.0 1.5

10 0 2* 30 3 0 116 8 90 3 010 0.2* 30 3.0 116 8 90 3.0

N = 13,000 k (last peak) = 5 column i.d. = 4. 6 mm mobile phase = 100


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*To achieve N= 13,000 on a 10 μm, 30 cm column, flow rate must be reduced to 0.2 mL/min

Comparison of Effect of Water/ACN and Water/MeOH on Viscosityy

1 80Viscosity (cP) - 25°C

1 201.401.601.80

0.600.801.001.20

cPMeOH 25°CACN 25°C

0.000.200.40 ACN 25°C

0 10 20 30 40 50 60 70 80 90 100

%B


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Comparison of Effect of Temperature on Viscosityy

1 8Viscosity (cP) - MeOH

1 21.41.61.8

MeOH 25^CMeOH 45^C

0 60.8

11.2

cP

0.20.40.6

%MeOH0

0 10 20 30 40 50 60 70 80 90 100


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Increasing Temperature Lowers Operating Pressurep g

Pressure, Analgesics Analysis, mobile phase: 15% acetonitrile, 4.6 x 50 mm, 1.8 um

400

300

350

200

250

ack

pres

sure

(bar

)

30C60C90C

100

150

Syst

em b

0

50

0 0 5 1 1 5 2 2 5 3 3 5 4 4 5


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0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Flow rate (mL/min.)

Higher Temperature as an Aid to Method Development and Faster Operation

Higher Temperature:

Temperature should always be consideredTemperature should always be considered as a parameter during method development

Decreases Mobile Phase ViscosityDecreases Mobile Phase ViscosityLowers backpressure – allows for higher flow rates, faster separations, greater efficiency and use of sub 2-micron columns

Provides more rapid mass transfer:Improves Efficiency – enhances resolutionDecreases analysis time faster separations with no loss in resolutionDecreases analysis time – faster separations with no loss in resolution

•Can change selectivity – optimize resolution


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Use Elevated Temperature to Optimize Resolution, Selectivity, y

50°C

Gradient of Ten Cardiac Drugs on SB-C18 RRHT

Rs=1.29

60°CRs=2.37

min0.5 1 1.5 2 2.5 3 3.5 4 4.5

70°C

Rs 2.37

min0.5 1 1.5 2 2.5 3 3.5 4 4.5

70°CRs=3.27


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min0.5 1 1.5 2 2.5 3 3.5 4 4.5

Gradient Retention (k*)

t F

Selectivity in gradient elution is determined by the gradient retention factor

t t tg F

S ΔΦ Vm

k* =k =t0

tR – t0

ΔΦ = change in volume fraction of B solventS = constantF = flow rate (mL/min.)tg = gradient time (min.)tg gradient time (min.)

Vm = column void volume (mL)

• S ≈ 4–5 for small molecules

• 10 < S < 1000 for peptides and proteins

In gradient separation the effective value of k (k*) for different bands will be about the same.


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`

g p ff f ( ) f ff

This Relationship Says that to Keep Relative Peak Position in the Chromatogram Unchanged

Column length Decrease in tG or F

Any Decrease in Can be Offset by a Proportional

Column length

Column volume (i d )

G

Increase in ΔΦ

Decrease in tG or F2Column volume (i.d.)

ΔΦ (same column)

Decrease in tG or F

Increase in ΔΦ

Decrease in tG or FΔΦ (same column) Decrease in tG or F

k* =tG • F


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k = S • ΔΦ • Vm

Two Chromatograms Both Having the Same Gradient Steepness

Sample: 1. Tebuthiuron 2. Prometon 3. Prometryne 4. Atrazine 5. Bentazon 6. Propazine 7. Propanil 8 Metolachlor

Gradient Steepness

4

5

Column: StableBond SB-C84.6 x 150 mm, 5 μm

GradientTime: 30 min.

45

Column: Rapid Resolution StableBond SB-C84.6 x 75 mm, 3.5 μm

Gradient

8. Metolachlor

Flow Rate: 1.0 mL/min

8

8

Time: 15 min.

Flow Rate: 1.0 mL/min

1

2

3AnalysisTime: 24 min 6

7

1

2

3

6

AnalysisTime: 12 min

0 5 10 15 20 25

min 77

Time (min)

0 5 10 15 20 25

Time (min)

0 5 10 15

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Presentation Objectives

Understand physical significance of chromatographic parameters especiallychromatographic parameters, especially Retention Factor and Resolution

Understand effect of role of Selectivity andUnderstand effect of role of Selectivity and Column Efficiency in improving Resolution

Column Selection

Parameters that affect column pressure


12/1/2009

SummaryHPLC is a powerful analytical toolThere are a variety of separation modes

Most applications are RPMost applications are RP

It’s important to know what the terms meanWe have introduced a number of chromatographic terms hereMany build up from simple measurements taken on a chromatogram

Key Equations Resolution

Many parameters can affect resolutionvan Deemter

Other things like pressure can affect our separation as wellOther things like pressure can affect our separation as wellDoes not have to be a limitation


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hplc separation fundamentals120109 - agilent separation fundamentals bill champion application...

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