(2001) Agilent - Fundamentals of Rf and Microwave Power Measurings
HPLC Separation Fundamentals120109 - Agilent Separation Fundamentals Bill Champion Application...
-
Upload
nguyenkhanh -
Category
Documents
-
view
224 -
download
0
Transcript of HPLC Separation Fundamentals120109 - Agilent Separation Fundamentals Bill Champion Application...
HPLC Separation pFundamentals
Bill ChampionApplication Engineer
Agilent Technologies, IncDecember 1, 2009
Separation fundamentalsAgilent Restricted
12/1/2009Page 1
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
12/1/2009Page 2
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
Separation fundamentals Agilent Restricted
12/1/2009Page 3
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
Separation fundamentals Agilent Restricted
12/1/2009
Volatile Volatility Nonvolatile
Page 4
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
Separation fundamentals Agilent Restricted
12/1/2009Page 5
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)
Separation fundamentals Agilent Restricted
12/1/2009Page 6
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
Separation fundamentals Agilent Restricted
12/1/2009Page 7
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)
Separation fundamentals Agilent Restricted
12/1/2009Page 8
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.
Separation fundamentals Agilent Restricted
12/1/2009
Time
Page 9
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
Separation fundamentals Agilent Restricted
12/1/2009Page 10
Chromatography Terms are All Around UsBut what do they meanBut what do they mean…..
Gradient Steepness
Peak Capacity
RRLC
UPLC
Separation fundamentals Agilent Restricted
12/1/2009Page 11
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)
Page 12 12/1/2009
Separation fundamentals Agilent Restricted
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
Page 13 12/1/2009
Separation fundamentals Agilent Restricted
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
Separation fundamentals Agilent Restricted
12/1/2009Page 14
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
Separation fundamentals Agilent Restricted
12/1/2009Page 15
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
Separation fundamentals Agilent Restricted
12/1/2009Page 16
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)
Page 17 12/1/2009
Separation fundamentals Agilent Restricted
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
Page 18 12/1/2009
Separation fundamentals Agilent Restricted
Chromatographic Terms Retention FactorRetention Factor
Two compounds can be separated if they have sufficiently different retention factors – k values
k =t0
tR – t0
tR2tR3tR1t00
Separation fundamentals Agilent Restricted
12/1/2009Page 19
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 ⟨
Separation fundamentals Agilent Restricted
12/1/2009Page 20
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
Separation fundamentals Agilent Restricted
12/1/2009
A Single Real Plate
Page 21
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
Page 22
k’: 2.0 4.5 7.0 9.5 12.0
12/1/2009
Separation fundamentals Agilent Restricted
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
Separation fundamentals Agilent Restricted
12/1/2009
1 2 3 4 5currently using.
Page 23
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
Separation fundamentals Agilent Restricted
12/1/2009
min0 1 2 3 4
Page 24
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)
Separation fundamentals Agilent Restricted
12/1/2009
reduce method development time (ChromSword, DryLab)
Page 25
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
Separation fundamentals Agilent Restricted
12/1/2009Page 26
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!!
Separation fundamentals Agilent Restricted
12/1/2009Page 27
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
Page 28 12/1/2009
Separation fundamentals Agilent Restricted
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
Separation fundamentals Agilent Restricted
12/1/2009Page 29
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
Separation fundamentals Agilent Restricted
12/1/2009
p g , g p gchromatographic resolution
Page 30
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
Separation fundamentals Agilent Restricted
12/1/2009
(mL/min)Smaller particle sizes yield flatter curves, minima shift to higher flow rates
Page 31
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
12/1/2009Page 32
Separation fundamentals Agilent Restricted
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
Separation fundamentals Agilent Restricted
12/1/2009Page 33
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
Separation fundamentals Agilent Restricted
12/1/2009Page 34
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.
Separation fundamentals Agilent Restricted
12/1/2009Page 35
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
Separation fundamentals Agilent Restricted
12/1/2009
*To achieve N= 13,000 on a 10 μm, 30 cm column, flow rate must be reduced to 0.2 mL/min
Page 36
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
Separation fundamentals Agilent Restricted
12/1/2009Page 37
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
Separation fundamentals Agilent Restricted
12/1/2009Page 38
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
Separation fundamentals Agilent Restricted
12/1/2009
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Flow rate (mL/min.)
Page 39
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
Separation fundamentals Agilent Restricted
12/1/2009Page 40
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
Separation fundamentals Agilent Restricted
12/1/2009
min0.5 1 1.5 2 2.5 3 3.5 4 4.5
Page 41
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.
Separation fundamentals Agilent Restricted
12/1/2009
`
g p ff f ( ) f ff
Page 42
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
Separation fundamentals Agilent Restricted
12/1/2009
k = S • ΔΦ • Vm
Page 43
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
12/1/2009Page 44
Separation fundamentals Agilent Restricted
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
Separation fundamentals Agilent Restricted
12/1/2009Page 45
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
Separation fundamentals Agilent Restricted
12/1/2009Page 46