Agilent HPLC PDF

Tips and Tricks of HPLC Separations
Agilent Technologies, Inc.

LC Tips And Tricks Seminar Series
April 12, 2011
Bill Champion
800-227-9770, opt 3, opt 3, op2
william_champion@agilent.com
Topics
• Chromatographic Process
• Improving Separations
• Troubleshooting

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Chromatographic Process
• Partition between mobile phase and
stationary phase
• Description of the separation:
Rs – Resolution
N – Column Efficiency, Plates
k, k’ – Retention Factor, Capacity Factor
α – Selectivity

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Some Basic Chromatography
Parameters
• Resolution (Rs)
• Retention Factor (k), Capacity Factor (k’)
• Selectivity or Separation Factor (α)
• Column Efficiency as
Theoretical Plates (N)

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Definition of Resolution
∆tR
Rs =
w
Resolution is a measure of the ability to separate

two components

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Definition of Resolution
tR-2 - tR-1 ∆tR

Rs = =
(w2 + w1)/2 w
Resolution is a measure of the ability to separate

two components

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Resolution …
Determined by 3 Key Parameters –
Efficiency, Selectivity and Retention
The Fundamental Resolution Equation
∆tR
=
w
N = Column Efficiency – Column length and particle size
α = Selectivity – Mobile phase and stationary phase
k = Retention Factor – Mobile phase strength

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Factors that Improve Resolution
…
. ∆tR
=
.
w
k Increase
retention
a
α Change relative
peak position
N
N Reduce peak
width

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Retention Factor (k), Capacity Factor (k’)
Chromatographic separation is an Equilibrium Process
Sample partitions between Stationary Phase and

Mobile Phase:
K = Cs/Cm
Compound moves through the column only while in

mobile phase.
Separation occurs in Column Volumes.

(Flow is volume/time – mL/min)

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K = Cs/Cm =>=> k = tR – t0
t0
k is measure of number of column volumes required to

elute compound.
Fundamental, dimensionless parameter that describes

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

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(VR – V0) (tR – t0)

k= =
V0 t0
Measure of number of column volumes required to

elute compound
Column Vol (V0)

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Un-retained component – elutes w/ solvent front

k=0
Column Vol (V0)

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Un-retained component – elutes w/ solvent front

k = (1 - 1) / 1 = 0
Column Vol (V0)

k = (tR – t0) / t0

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Component retained – elutes in 1 add’l column volumes

k = (2 - 1) / 1 = 1
1 2
Column Vol (V0)

k = (tR – t0) / t0

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k = (3 - 1) / 1 = 2
1 2 3
Column Vol (V0)

k = (tR – t0) / t0

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k = (8 - 1) / 1 = 7
1 2 3 4 5 6 7 8
Column Vol (V0)

k = (tR – t0) / t0

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Chromatographic Profile
Equations Describing Factors Controlling RS
Retention Factor
(tR-t0)
k=
t0
Selectivity
α = k2/k1
Theoretical Plates-Efficiency
N = 16(tR / tW)2

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Retention Factor
(tR-t0)
k=
t0
Selectivity
α = k2/k1
N = 16(tR / tW)2
k = 1 to 20 - OK; k = 3 to 10 - Better; k = 5 to 7 - Ideal

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Retention Factor
(tR-t0)
k=
t0
Selectivity
α = k2/k1
N = 16(tR / tW)2

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Selectivity (α)
α = k2
k1
α is measure relative difference in retention

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Selectivity (α)
k2
α = = (tR2 – t0)/t0
k1 (tR1 – t0)/t0

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Selectivity (α)
k2
α = = (tR2 – t0)
k1 (tR1 – t0)

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Selectivity (α)
α = k2
k1
α is measure of relative difference in retention
By definition, k2 is more retained component;

k1 is less retained component, so α is always ≥ 1
To obtain separation, α must be > 1

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Retention Factor
(tR-t0)
k=
t0
Selectivity
α = k2/k1
Theoretical Plates - Efficiency

N = 16(tR / tW)2

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Retention Factor
(tR-t0)
k=
t0
tR
Selectivity
α = k2/k1
tW0.5
Theoretical Plates - Efficiency
N = 16(tR / tW)2
N = 5.54(tR / tW0.5)2

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Column Efficiency (N)
N - Number of theoretical plates.
“Plates” is a term inherited from distillation theory. It is a measure

of the relative peak broadening (or peak width) for an analyte in a
separation – w
tR 2
N = 16 w
A Number of Theoretical Plates

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Column Efficiency (N)
N - Number of theoretical plates.
We can increase N by increasing the length of the column or

decreasing the size of the stationary phase particles.
(1.8 µm > 3.5 µm > 5 µm > 10 µm)
tR 2
N = 16 w = f(L, 1/dp)
L = column length
dp = particle size

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Van Deemter Curve
Plate Height H (L/N)
Factors Affecting N
H = L/N Large Small

Particle Particle
H = A + B/u + C u
Resistance to Mass Transfer

Linear Velocity u
The smaller the plate height, the higher the plate number and the greater the
chromatographic resolution
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Van Deemter Curve
Effect of Particle Size
H = A + B/u + Cu
Column: ZORBAX Eclipse XDB-C18
0.0030
Dimensions: 4.6 x 50/30mm
Eluent: 85:15 ACN:Water
0.0025 Flow Rates: 0.05 – 5.0 mL/min
Temp: 20°C
µL Octanophenone in Eluent 5.0 µm
HETP (cm)
Sample: 1.0µ
0.0020
µm
5.0µ
0.0015
µm
3.5µ
µm
1.8µ 3.5 µm
0.0010
0.0005 1.8 µm
0.0000
0.0 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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Columns Packed with Smaller Particles
Provide Higher Efficiency
sub 2 micron
3 micron Large Small

Particle Particle
N N α 1/(dp)
5 micron
P α 1/(dp)2
10 micron
Velocity
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Topics
• Troubleshooting

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Improving the Separations
• Improve Selectivity (α)
• Improve Column Efficiency (N)
• Improve Chromatography Choices

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Selectivity
• Mobile Phase
• Stationary Phase

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Different Mobile Phases May
Give Different Selectivity
mAU
700 O
600
50/50 MeOH/HOH Me
500
... Anisole
400
Phenethanol ...
300
200 p-F-Phenethanol ….....

100 Toluene ...
0
0 2 4 6 8 10 12 14 16 18 min
mAU
700
600 41/59 ACN/HOH

500
OH
400
300
200
100
0 2 4 6 8 10 12 14 16 18 min
ZORBAX® SB-C18 4.6 x 250 mm

1 mL/min, 40°C, 225 nm

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Effect of pH on Retention
aa

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Effect of pH on Retention, Peak Shape

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4/13/2011
Test for pH Robustness
Column: ZORBAX Rapid Resolution Eclipse XDB-C8, 4.6 x 75 mm, 3.5 µm
Mobile Phase: 44% 25 mM phosphate, pH 7.00 : 56% methanol Flow Rate: 1.0 mL/min Temperature: 25°C
Detection: UV 250 nm
Sample: 1. ketoprofen 2. ethyl paraben 3. hydrocortisone 4. fenoprofen 5. propyl paraben 6. propranolol 7. ibuprofen
1 4 5
2
pH 7.00 7
6
6
7
0 1 2 3 4 5 6
6 Time (min)
pH 7.25
1 2 4
5
6,7 6 7
7 0 1 2 3 4 5 6
Time (min)
• The resolution of ionizable compounds can change

markedly with pH changes—even as small as 0.05–
0.25 pH units.
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4/13/2011
Effect of pH on Peak Shape at
or Near the Sample pKa
Column: ZORBAX SB-C8 4.6 x 150 mm, 5 mm
Mobile Phase: 40% 5 mM KH2PO4: 60% ACN
Flow Rate: 1.0 mL/min
Temperature: RT
CH3CHCOOH
pH 4.4 pH 3.0
CH2CH(CH3)2
Ibuprofen
pKa = 4.4
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10
Time (min) Time (min)
• Inconsistent and tailing peaks may occur when operating close to an analyte’s
pKa and should be avoided.

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Different Stationary Phases May
Give Significantly Different Selectivity
Columns: Mobile Phase:
mAU
Zorbax StableBond, 3.0 X 100 mm, 3.5-mm 6 38% methanol in 0.4% formic acid OH OH
300
Zorbax SB-CN HO O HO O
200 5 OH OH
3 4 7
OH OH
100 1 2 OH OH
(+)-Catechin (-)-Epicatechin
0
0 2 4 6 8 10OH 12 min
mAU OH
O O O
HO HO O
6
OH
300
Zorbax SB-AQ HO
OH
O
OH O OH O
O
OH
200 O
OH O
H3C O O
1 2 3 4 5
OH
H3C OH
100 OH
7 OH
Naringin
HO
OH
OH
Rutin
0
0 2 4 6 8 OH 10 12 min*
mAU
300
6 OH OH
Zorbax SB-Phenyl 3
HO O HO O
200
7 5 OH
100
1 2 4 OH O OH O
Quercetin 4',5,7-Trihydroxyflavanone
0
0 2 4 6 8 10 12 min*
mAU CH3
300 Zorbax SB-C18

200
6
OH
Flavanones
100 1 2 3 4 7 5 H3C CH3
Thymol
2 4 6 8 10 12 min
*
0
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Similar Stationary Phases May Give
Different Selectivity
1 2 4 Eclipse Plus C18
1st choice 3
Best Resolution & Peak Shape Mobile phase: (69:31) ACN: water
Flow 1.5 mL/min.
1 2 3 4 5 Temp: 30 °C
2nd choice Detector: Single Quad ESI
Good alternate selectivity 1 2 4 StableBond positive mode scan
3 Columns: RRHT
due to non-endcapped SB-C18 4.6 x 50 mm 1.8 um
1 2 3 4 5 min Sample:
1. anandamide (AEA)
3rd choice 1 4 Eclipse 2. Palmitoylethanolamide (PEA)
2
Good efficiency & peak shape XDB-C18 3. 2-arachinoylglycerol (2-AG)
3
Resolution could be achieved 4. Oleoylethanolamide (OEA)
1 2 3 4 5
Multiple bonded
4th choice phases for most
Resolution not likely, Extend-C18 effective method
1 2,3
Other choices better, for this 4
separation.
development.
Match to one you’re
1 2 3 4 5
currently using.
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Columns Packed with Smaller Particles
Provide Higher Efficiency
sub 2 micron
3 micron
N α 1/(dp)
5 micron
P α 1/(dp)2
10 micron
Velocity
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Decreasing Particle Size
5 µm
8 min, 170 bar
3.5 µm
6 min, 204 bar
1.8 µm 3.4 min, 300 bar

“Reversed-Phase HPLC Separation of
Water-Soluble Vitamins on Agilent
ZORBAX Eclipse Plus Columns”,
5989-9313EN (2008)

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Improve Chromatography Choices
• Shorten analysis time:
reduce column length,
increase flow rate
• Sample Preparation

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increase flow rate

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Reduce analysis time
• 250 mm, 5 um ~ 150 mm, 3.5 um – 60%
• 2 mL/min vs 1 mL/min – 50%
• Reduce 25 min run to 7.5 min run

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USP Method for Naproxen Tablets – 4X Faster
Analysis on Poroshell 120
Method Requirement N > 4000, Rs better than 11.5
4.6 x 150 mm (L1) Common Conditions:
Eclipse Plus C18, 5um Mobile Phase: 50:49:1 MeCN:H2O Acetic Acid
mAU
30
PN 959993-902 Flow Rate: 1.2 mL/min
25 20 ul injection Peak 1. Naproxen 2. Butyrophenone
20
15
Rs = 14.9 N= 12,554
10
5 N= 14,885
0
1 2 3 4 5 6 7 8 9 min
4.6 x 100 mm (L1)
mAU
30 N= 21,046 Poroshell 120 EC-C18, 2.7 um
25 PN 695975-902
20
N= 20,676 13.67 ul injection 2X Faster
15
10
Rs = 17.0
5 P = 238 bar
0
1 2 3 4 5 6 7
4.6 x 50 mm (L1)
mAU
30 N= 11,281 Poroshell 120 EC-C18, 2.7 um
25
20 N= 12,051
PN 699975-902 4X Faster
15
6.7 ul injection
10 Rs = 12.6
5
0
P= 133 bar
1 2 3 4 5 6 7 min

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increase flow rate

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Split Peaks from
Injection Solvent Effects
Column: StableBond SB-C8, 4.6 x 150 mm, 5 µm; Mobile Phase: 82% H2O : 18% ACN; Inj Vol: 30 µL
Sample: 1. Caffeine 2. Salicylamide
1
A. Injection Solvent B. Injection Solvent

100% Acetonitrile Mobile Phase
2
2
0 10 0 10
Tip: Injecting in a solvent stronger than the mobile phase can cause peak
shape problems such as peak splitting or broadening
Trick: Keep Organic Concentration in Sample Solvent < Mobile Phase

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Columns Die from the Sample
Prevention Techniques - A Better Choice!
• Use column protection

- In-line filters
- Guard columns Easy
• Filter samples
• Filter buffered mobile phases
• Sample clean-up (i.e. SPE)

Not as Easy
• Appropriate column flushing
Column cleaning: R. Majors, LCGC (2003) Vol 21 p19.

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Column Cleaning
Flush with stronger solvents than your mobile phase
Reversed Phase Solvent Choices
In Order of Increasing Strength
Use at least 25 mL of each solvent for analytical
columns
• Mobile phase without buffer salts
• 100% Methanol
• 100% Acetonitrile
This Is time consuming • 75% Acetonitrile:25% IPA Must reverse
Often performed offline • 100% Isopropanol
to
• 100% Methylene Chloride*
• 100% Hexane* Re-equilibrate
Tip: When using either Hexane or Methylene Chloride; The column must be flushed
with Isopropanol before returning to your reverse phase mobile phase.
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Topics
• Troubleshooting – Poor Peak Shape

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Peak Tailing, Broadening
and Loss of Efficiency
May be caused by:
• Column “secondary interactions”

• Column contamination
• Column aging
• Column loading
• Extra-column effects

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Split Peaks from
Column Contamination
Column: StableBond SB-C8, 4.6 x 150 mm, 5 µm Mobile Phase: 60% 25 mM Na2HPO4, pH 3.0 : 40% MeOH Flow Rate: 1.0 mL/min
Temperature: 35°C Detection: UV 254 nm Sample: Filtered OTC Cold Medication: 1. Pseudoephedrine 2. APAP 3. Unknown 4. Chlorpheniramine
Injection 1
Injection 1 Injection 30
After Column Wash
with 100% ACN
2
2 2
4
1
1
4
1 3
4
3
3
0 5 10 15 0 5 10 15
0 5 10 15 Time (min)
Tip: Column washing eliminates the peak splitting, which resulted from a contaminant on the column
How could this be prevented? (Guard Column, SPE clean up of samples, Periodic column wash)

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Peak Splitting Caused By Disrupted Sample Path
• Flow path disrupted by void
• Sample allowed to follow different
..paths through column
• Poorly packed bed settles in use
• High pH dissolves silica
Normal Double
Peaks
Tip: Similar Effect Can be Caused by Partially Plugged Frit

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Peak Shape: Tailing Peaks
Symmetry > 1.2 Causes

Some Peaks Tail
Secondary - Retention Effects.
Residual Silanol Interactions.
Small Peak Eluting on Tail of Larger Peak.
Normal Tailing
All Peaks Tail

Extra-Column Effects.
Build up of Contamination on
Column Inlet.
Heavy Metals.
Normal Tailing Bad Column.

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Peak Tailing - Column Contamination
Tip: Quick test to determine if column is dirty or damaged
Trick: Reverse column and run sample
• If improved; Possible cleaning will help
• No improvement; Column damaged and needs to be replaced
QC test forward QC test reverse direction QC test after cleaning

direction 100% IPA, 35°C
Plates Tf 3
Plates Tf 3
Plates Tf
3
1. 7629 2.08 1. 7448 1.06
1. 7906 1.43
2. 12043 1.64 2. 12237 1.21
2. 12443 1.21
3. 13727 1.69 3. 15366 1.11
3. 17999 1.19
4 13355 1.32 2 4 19067 1.17 2
4 17098 1.25
2
4
1
4
1 1 4
0.0 2.5 5.0 0.0 2.5 5.0 0.0 2.5 5.0

Time (min) Time (min) Time (min)
Column: StableBond SB-C8, 4.6 x 250 mm, 5µm Mobile Phase: 20% H2O : 80% MeOH Flow Rate: 1.0 mL/min
Temperature: R.T. Detection: UV 254 nm Sample: 1. Uracil 2. Phenol 3. 4-Chloronitrobenzene 4. Toluene

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Why Worry About pH?
pH, pKa and Weak Bases
R3NH+ R 3N + H+ [R3N][H+]
Ka =
[R3NH+]
∅
+ Ka = 1 x 10-9
CH 3
∅ CH 3
H pKa = 9
∅
CHOCH CH N
2 2
CHOCH CH N
2 2
+ H+
CH ∅ CH
3 3
At pH 9 – the sample exists as protonated and unprotonated diphenhydramine

in a ratio of 1:1. Peak shape can be poor.
At pH 10 – 91% of the sample exists as unprotonated diphenhydramine.
At pH 8 – 91% of the sample exists as protonated diphenhydramine.

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The Surface of Silica Supports for HPLC
OH HO OH OH OH
Si Si Si Si
Free Geminol Associated

Silanols Silanols Silanols
decreasing acidity
OH
+ +
M M Si
Surface Metal Internal Metal
(activated silanol)
(most acidic)

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Choose the Best Bonded-Phase
for Each pH Range
Eclipse Plus, Eclipse XDB, pH 2-9

Low and Mid pH
1. Densely Bonded Extend-C18, pH 2-11.5
dimethylalkylsilanes Bonus-RP, pH 2-8
StableBond, pH 1-6 2. proprietary double-endcapping Use at Low and Mid pH Use at High pH
Use at Low pH 1. unique bidentate structure
CH3 1. polar alkyl phase
1. Uses bulky silanes 2. double endcapped
O Si 2. triple endcapped
2. Non-endcapped CH3 3. uses bulky silanes
CH3 C18 C18
O Si CH3 R1
* R
* CH3
O Si PG R
Si Si
CH3
O O
O Si R1 O Si R1
CH3 Silica Support
CH3
R CH3
Si CH3
OH O Si CH3
R CH3
CH3
R1
CH3
O Si R1 O Si O Si PG R
CH3
R R1
OH CH3
R Si CH3
CH3
O Si R1 R1
R O Si PG R
R1

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Peak Tailing
Low pH Minimizes “Secondary Interactions” for Amines
Column: Alkyl-C8, 4.6 x 150 mm, 5µm Mobile Phase: 85% 25 mM Na2HPO4 : 15% ACN Flow Rate: 1.0 mL/min
Temperature: 35°C Sample: 1. Phenylpropa nolamine 2. Ephedrine 3. Amphetamine 4. Methamphetamine 5. Phenteramine
1
3
2
pH 3.0 pH 7.0
USP Tf (5%) 4
2 3 USP Tf (5%)
5
4. 1.33 4. 2.35
4
5
0.0 2.5 5.0 0.0 2.5 5.0

Tip: Reducing mobile phase pH reduces silanol interaction and peak tailing.

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Peak Tailing
High pH Eliminates “Secondary Interactions” for Amines
Column: ZORBAX Extend-C18, 4.6 x 150 mm, 5 m m
Mobile Phase: See Below , Flow Rate: 1.0 mL/min , Temperature: RT, Detection: UV 254 nm
Sample: 1. Maleate 2. Scopolamine 3. Pseudoephedrine 4. Doxylamine 5. Chlorpheniramine 6. Triprolidine 7. Diphenhydramine
pH 7 pH 11
30% 20 mM Na2HPO4 30% 20 mM TEA
70% MeOH 70% MeOH
7
4 7
4
2,3
3
1
tR = 8.5 1
5 tR = 11.4
5
6 2
6
0 5 0 5 10
Peak shape and retention of this sample of basic compounds improves

at high pH where column has high IEX activity. Why?

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Peak Tailing
Identifying Column “Secondary Interactions”
Column: Alkyl-C8, 4.6 x 150 mm, 5µm Mobile Phase: 85% 25 mM Na2HPO4 pH 7.0 : 15% ACN Flow Rate: 1.0 mL/min
Temperature: 35°C Sample: 1. Phenylpropa nolamine 2. Ephedrine 3. Amphetamine 4. Methamphetamine 5. Phenteramine
1 1
3
2
No TEA
2 3 10 mM TEA
USP Tf (5%) 4 USP TF (5%)
5
1. 1.29
4
5 1. 1.19
2. 1.91 2. 1.18
3. 1.63 3. 1.20
4. 2.35 4. 1.26
5. 1.57 5. 1.14
0.0 2.5 5.0 0.0 2.5 5.0

TIme (min) Time (min)
Tip: Mobile phase modifier (TEA) competes with sample for surface ion exchange
sites at mid-range pH values
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Column Connectors Used in HPLC
Swagelok Waters
0.090 0.130
in. in.
Parker Rheodyne
0.170
0.090
in.
in.
Valco Uptight
0.080 0.090
in. in.
Troubleshooting LC Fittings, Part II. J. W. Dolan and P. Upchurch, LC/GC Magazine 6:788 (1988)

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What Happens If Connections Are Poorly Made?
Wrong … too long

Ferrule cannot seat properly
Wrong … too short

X Mixing Chamber
If Dimension X is too long, leaks will occur
If Dimension X is too short, a dead-volume,

or mixing chamber, will occur

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Topics
Separation occurs in column volumes
Selectivity
Column efficiency
Control pH
• Troubleshooting
Sample clean-up
Secondary interaction
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Thank you – Questions?
Bill Champion
800-227-9770, opt 3, opt 3, op2
william_champion@agilent.com

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Retention vs. pH for Ionizable Compounds
Effects are Compound Dependent
Mobile Phase:
12 45% MeOH:
55% 20 mM Phosphate Buffer
10
retention time (min)
6 Acidic
4
2 Neutral
0
pH 2.5 pH 6.5 pH 8 pH 11.5
pH

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Page 69 4/13/2011

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Tips and Tricks of HPLC Separations

Agilent Technologies, Inc.

LC Tips And Tricks Seminar Series

LC Tips And Tricks Seminar Series

LC Tips And Tricks Seminar Series

Resolution is a measure of the ability to separate

LC Tips And Tricks Seminar Series

tR-2 - tR-1 ∆tR

Resolution is a measure of the ability to separate

LC Tips And Tricks Seminar Series

N = Column Efficiency – Column length and particle size

α = Selectivity – Mobile phase and stationary phase

k = Retention Factor – Mobile phase strength

LC Tips And Tricks Seminar Series

LC Tips And Tricks Seminar Series

Sample partitions between Stationary Phase and

Compound moves through the column only while in

Separation occurs in Column Volumes.

LC Tips And Tricks Seminar Series

k is measure of number of column volumes required to

Fundamental, dimensionless parameter that describes

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

LC Tips And Tricks Seminar Series

(VR – V0) (tR – t0)

Measure of number of column volumes required to

Column Vol (V0)

LC Tips And Tricks Seminar Series

Un-retained component – elutes w/ solvent front

Column Vol (V0)

LC Tips And Tricks Seminar Series

Un-retained component – elutes w/ solvent front

Column Vol (V0)

LC Tips And Tricks Seminar Series

Component retained – elutes in 1 add’l column volumes

Column Vol (V0)

LC Tips And Tricks Seminar Series

Component retained – elutes in 2 add’l column volumes

Column Vol (V0)

LC Tips And Tricks Seminar Series

Component retained – elutes in 7 add’l column volumes

Column Vol (V0)

LC Tips And Tricks Seminar Series

LC Tips And Tricks Seminar Series

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

LC Tips And Tricks Seminar Series

α is measure relative difference in retention

LC Tips And Tricks Seminar Series

α is measure relative difference in retention

LC Tips And Tricks Seminar Series

α is measure relative difference in retention

LC Tips And Tricks Seminar Series

α is measure of relative difference in retention

By definition, k2 is more retained component;

To obtain separation, α must be > 1

LC Tips And Tricks Seminar Series

Theoretical Plates - Efficiency

LC Tips And Tricks Seminar Series

LC Tips And Tricks Seminar Series

N - Number of theoretical plates.

“Plates” is a term inherited from distillation theory. It is a measure

A Number of Theoretical Plates

LC Tips And Tricks Seminar Series

N - Number of theoretical plates.