General Principles of HPLC Method Development
General Principles of HPLC Method Development
General Principles of HPLC Method Development
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<strong>General</strong> <strong>Principles</strong><br />
<strong>of</strong> <strong>HPLC</strong> <strong>Method</strong><br />
<strong>Development</strong><br />
Philip J. Koerner, Ph.D.<br />
Thailand<br />
September 2013<br />
Part 1. <strong>General</strong> Chromatographic Theory<br />
Part 2. The Stationary Phase:<br />
An Overview <strong>of</strong> <strong>HPLC</strong> Media<br />
Part 3. Role <strong>of</strong> the Mobile Phase in Selectivity
The Liquid Chromatographic Process<br />
3<br />
Column Chromatography:<br />
The Beginning <strong>of</strong> Liquid<br />
Chromatography<br />
Empty<br />
Column<br />
Adsorbent<br />
particles<br />
added<br />
Sample is<br />
loaded onto<br />
the top <strong>of</strong><br />
the column<br />
Solvent is<br />
added to the<br />
top <strong>of</strong> the<br />
column<br />
Separation<br />
occurs as<br />
the bands<br />
move down<br />
the column<br />
4<br />
Mikhail Tsvet, 1872-1919
Basis <strong>of</strong><br />
Chromatographic Separation<br />
Separation <strong>of</strong> compounds by <strong>HPLC</strong> depends on the interaction <strong>of</strong><br />
analyte molecules with the stationary phase and the mobile phase.<br />
Mobile Phase<br />
Stationary Phase<br />
5<br />
The Liquid<br />
Chromatographic Process<br />
Analytes<br />
Mobile Phase<br />
Stationary Phase<br />
6
The Liquid<br />
Chromatographic Process<br />
Analytes<br />
Mobile Phase<br />
Stationary Phase<br />
7<br />
The Liquid<br />
Chromatographic Process<br />
Polar/Aqueous<br />
Mobile Phase<br />
H<br />
O<br />
N<br />
O H<br />
H<br />
N<br />
N<br />
H<br />
O H<br />
N<br />
O H<br />
H<br />
N<br />
O H<br />
H<br />
H<br />
Benzo(a)pyrene<br />
Ethyl sulfate<br />
Non-Polar Stationary<br />
Phase (e.g. C18)<br />
8
In any separation, almost never get a pure, single mode <strong>of</strong> separation. In RP,<br />
performance will be dictated by mixture <strong>of</strong>:<br />
1. Hydrophobic interactions<br />
2. Polar interactions<br />
3. Ionic interactions<br />
Mechanisms <strong>of</strong> Interaction<br />
In RP Chromatography<br />
<strong>Method</strong> <strong>Development</strong> = modulating stationary phase and mobile phase<br />
conditions to optimize these interactions and achieve a specific separation<br />
goal.<br />
OH<br />
Tapentadol<br />
CH 3<br />
CH 3<br />
N<br />
9<br />
CH 3<br />
CH 3<br />
Hydrophobic Interactions<br />
Hydrophobic<br />
C H 3<br />
C H 3 C H 3 C<br />
OH<br />
• Weak, transient interactions<br />
between a non-polar stationary<br />
phase and the molecules<br />
O<br />
C H 3<br />
H 3<br />
C H 3<br />
• Hydrophobic & van Der<br />
Waals interactions<br />
CH 3<br />
CH 3<br />
N<br />
H3C<br />
CH 3<br />
• Retention will be predicted by<br />
Log P values<br />
S i H 3 C S i<br />
H C S i<br />
H 3 C<br />
S i<br />
C H 3<br />
O C H 3<br />
C H 3<br />
O 3<br />
O - O H C H O H<br />
O O 3<br />
O<br />
C H 3<br />
S i<br />
C H 3<br />
C H 3<br />
S i S i S i S i S i S i S i<br />
O O O O O O<br />
H O O H H O O H O H O H H O<br />
O<br />
S i S i S i<br />
O O O H<br />
O H O H O H<br />
10
Polar Interactions<br />
C H 3<br />
C H 3 C H 3 C<br />
Polar<br />
• Interactions between polar<br />
functions groups <strong>of</strong> analyte<br />
and residual silanols or polar<br />
groups on media<br />
O<br />
C H 3<br />
H 3<br />
C H 3<br />
CH<br />
H C 3<br />
3<br />
N<br />
H 3 C<br />
H 3 C<br />
• Hydrogen bonding, dipoledipole<br />
interactions<br />
• Relatively weak and transient<br />
HO<br />
S i H 3 C S i<br />
H C S i<br />
H 3 C<br />
S i<br />
C H 3<br />
O C H 3<br />
C H 3<br />
O 3<br />
O - O H C H O H<br />
O O 3<br />
O<br />
C H 3<br />
S i<br />
C H 3<br />
C H 3<br />
S i S i S i S i S i S i S i<br />
O O O O O O<br />
H O O H H O O H O H O H H O<br />
O<br />
S i S i S i<br />
O O O H<br />
O H O H O H<br />
11<br />
Ion-exchange Interactions<br />
• Interactions between<br />
ionizable functional groups on<br />
analyte and counter-charged<br />
moiety on stationary phase<br />
Ion-Exchange<br />
• Ion-exchange<br />
C H 3<br />
C H 3 C H 3 C<br />
OH<br />
• Strong, slow interactions<br />
O<br />
C H 3<br />
H 3<br />
C H 3<br />
CH 3<br />
H 3 C<br />
H N<br />
+<br />
H<br />
S i S i<br />
3 C<br />
H C<br />
CH 3<br />
3 H C S i<br />
H 3 C<br />
S i<br />
C H 3<br />
O C H 3<br />
C H 3<br />
O 3<br />
O - O H C H O H<br />
O O 3<br />
O<br />
C H 3<br />
S i<br />
C H 3<br />
C H 3<br />
S i S i S i S i S i S i S i<br />
O O O O O O<br />
H O O H H O O H O H O H H O<br />
O<br />
S i S i S i<br />
O O O H<br />
O H O H O H<br />
12
Chromatographic Measurements<br />
13<br />
Chromatographic<br />
Measurements<br />
14<br />
k’ Asym N α
Void Volume<br />
Analytes which do not interact with the adsorbent elute from the column in a<br />
volume equal to the void volume in the column. The void volume <strong>of</strong> a<br />
column is the amount <strong>of</strong> mobile phase in the column between the adsorbent<br />
particles and in the pores <strong>of</strong> the porous adsorbent particles.<br />
Mobile phase occupies the space<br />
between the particles or the<br />
interstitial volume.<br />
Mobile phase fills the pores <strong>of</strong> the<br />
porous adsorbent particles.<br />
15<br />
Void Volume<br />
A compound which does not interact with the adsorbent at all elutes at<br />
what is termed the void volume or the solvent front. The time that it<br />
takes for non-retained components to elute is the void time or t 0<br />
.<br />
• void volume<br />
• solvent front<br />
• t 0<br />
16
Retention Factor (k’)<br />
The retention factor <strong>of</strong> the eluting compound is its elution volume (time)<br />
relative to the elution volume (time) <strong>of</strong> an unretained compound. The k’<br />
value for a given analytes will be determined by its relative affinity for the<br />
stationary phase and mobile phase.<br />
Retention factor (k’)=<br />
t R – t 0<br />
t 0<br />
t 0<br />
17<br />
Retention Factor (k’)<br />
For any given analyte, the k’ value will be most readily modified by changing<br />
the % <strong>of</strong> strong mobile phase (e.g. methanol or ACN).<br />
Example: The Separation <strong>of</strong> Steroids:<br />
Column used: Phenomenex Luna C18(2) 150 x 4.6 mm<br />
18
Retention Factor (k’):<br />
Effect <strong>of</strong> Changing % ACN<br />
19<br />
Peak Asymmetry (Asym)<br />
The asymmetry value (Asym) for a peak is a measure <strong>of</strong> the divergence <strong>of</strong><br />
the peak from a perfect Gaussian peak shape. Peaks with asymmetry<br />
values > 1.0 are said to be “tailing”, those with asymmetry values < 1.0 are<br />
said to be “fronting”.<br />
Asymmetrical peaks can be<br />
attributed to a number <strong>of</strong> factors:<br />
(1) Strong secondary interactions (e.g.<br />
ionic interactions between basic<br />
compounds and residual silanols)<br />
(2) Sample overloading<br />
(3) Sample solvent incompatibility<br />
(4) Poor packing<br />
20
Peak Tailing due to<br />
Secondary Interactions<br />
Classical peak tailing in reversed-phase methods is most commonly caused<br />
by strong ionic interactions between basic analytes and residual silanols<br />
on the surface <strong>of</strong> the silica.<br />
C H 3<br />
C H 3 C H 3<br />
C<br />
O<br />
C H 3<br />
H 3<br />
C H 3<br />
Ion-Exchange<br />
O H<br />
C H 3<br />
H 3 C<br />
H 3 C<br />
H N<br />
+<br />
C H 3<br />
S i H S i 3 C<br />
S i S i<br />
O C H 3<br />
C H<br />
O 3<br />
O - O H H 3 C<br />
H C C H 3 3<br />
C H O H<br />
O O 3<br />
O<br />
C H 3<br />
S i<br />
C H 3<br />
C H 3<br />
S i S i S i S i S i S i S i<br />
O O O O O O<br />
H O O H H O O H O H O H H O<br />
O<br />
S i S i S i<br />
O O O H<br />
O H O H O H<br />
21<br />
Peak Tailing due to<br />
Secondary Interactions<br />
Example: The Separation <strong>of</strong> Tricyclic Antidepressants:<br />
Column used: Kinetex 2.6µ XB-C18 50 x 2.1 mm<br />
Brand H 2.7µ C18 50 x 2.1mm<br />
Amitriptyline (pKa 9.4) Nortriptyline (pKa 9.7) Protriptyline (pKa 8.2)<br />
22
30<br />
25<br />
20<br />
15<br />
10<br />
5<br />
50<br />
40<br />
30<br />
20<br />
10<br />
100<br />
80<br />
60<br />
40<br />
20<br />
DAD1 C, Sig=254,4 Ref=300,100 (DJ040611\DJGSK 2011-04-06 12-28-48\GSK000006.D)<br />
12 12.5 13 13.5 14 14.5 15 15.5 16 16.5<br />
DAD1 C, Sig=254,4 Ref=300,100 (DJ040611\DJGSK 2011-04-06 12-28-48\GSK000008.D)<br />
12.5 13 13.5 14 14.5 15 15.5 16 16.5<br />
DAD1 C, Sig=254,4 Ref=300,100 (DJ040611\DJGSK 2011-04-06 12-28-48\GSK000010.D)<br />
12.5 13 13.5 14 14.5 15 15.5 16 16.5<br />
min<br />
min<br />
min<br />
Peak Tailing due to<br />
Secondary Interactions<br />
Brand H 2.7µ C18 50x2.1mm<br />
Kinetex 2.6µ XB-C18 50x2.1mm<br />
XIC <strong>of</strong> +MRM (8 pairs): 264.2/191.1 Da ID: Protrip from Sample 28 (Halo C18-50x2, 2.7 um, MeOH:0.1% ... XIC <strong>of</strong> +MRM (8 pairs): 278.2/191.2 Max. Da 1.1e5 ID: Amitrip cps. from Sample 7 (TCA-Kin-XB-C18, 50x2, 2.6, MeOH 0....<br />
1 5<br />
5<br />
5<br />
4<br />
1<br />
1<br />
4<br />
Peak tailing<br />
Max. 1.2e5 cps.<br />
6<br />
6<br />
2 3<br />
7 8<br />
2 3<br />
7 8<br />
3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0<br />
Time, min<br />
Time, min<br />
XIC <strong>of</strong> +MRM (8 pairs): 264.2/191.1 Da ID: Protrip from Sample 28 (Halo C18-50x2, 2.7 um, MeOH:0.1% ... XIC <strong>of</strong> +MRM (8 pairs): 264.2/191.1 Max. Da 1.1e5 ID: Protrip cps. from Sample 13 (Kinetex XB-C18-50x2, 2.6 um, MeO...<br />
Max. 1.2e5 cps.<br />
4<br />
4<br />
6<br />
6<br />
3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0<br />
Time, min<br />
3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0<br />
Time, min<br />
23<br />
Mobile phase: A = 0.1% Formic acid in water, B = 100% Methanol,<br />
Gradient: 15 to 60% B in 5 min, hold for 1 min<br />
Flow rate: 400 µL/min<br />
1. Amoxapine<br />
2. Imipramine<br />
3. Desipramine<br />
4. Protriptyline*<br />
5. Amitriptyline<br />
6. Nortriptyline*<br />
7. Clomipramine<br />
8. Norclomipramine<br />
Peak Tailing due to<br />
Sample Overloading<br />
Strongly basic analytes are very sensitive to sample loading, and will display<br />
peak tailing effects as a function <strong>of</strong> increasing loading (µg on column):<br />
mAU0<br />
2.5 µg on column;<br />
USP Tailing = 1.17<br />
14.208<br />
pKa 9.7<br />
5 µg on column;<br />
USP Tailing = 1.34<br />
14.169<br />
mAU0<br />
12<br />
12 µg on column;<br />
USP Tailing = 1.87<br />
14.157<br />
mAU0<br />
12<br />
24
1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7<br />
25<br />
min<br />
Peak Fronting due to<br />
Sample Overloading<br />
Neutral and acidic compounds will typical show peak fronting when the<br />
column is overloaded.<br />
Detector saturation!<br />
DAD1 A, Sig=240,10 Ref=<strong>of</strong>f (MT042211\DJGSK 2011-04-22 09-12-57\MUPIROCIN000001.D)<br />
2250<br />
2.495<br />
2000<br />
1750<br />
1500<br />
mAU<br />
Fronting due to overload<br />
1250<br />
1000<br />
750<br />
500<br />
250<br />
1.905<br />
1.745 0<br />
2.145<br />
Peak Fronting due to<br />
Sample Solvent Effects<br />
Peak fronting can also be due to sample solvent effects:<br />
(1) Sample insolubility<br />
(2) Sample solvent is too strong:<br />
Sample in 50% Methanol<br />
7.0e4<br />
6.5e4<br />
6.0e4<br />
5.5e4<br />
5.0e4<br />
4.5e4<br />
Hydromorphone<br />
Sample in 100% Methanol<br />
2.2e5<br />
2.0e5<br />
1.8e5<br />
1.6e5<br />
Breakthrough!<br />
1.4e5<br />
4.0e4<br />
3.5e4<br />
3.0e4<br />
1.2e5<br />
1.0e5<br />
0.24<br />
Fronting<br />
2.5e4<br />
8.0e4<br />
2.0e4<br />
1.5e4<br />
1.0e4<br />
5000.0<br />
0.0<br />
Morphine<br />
1.06<br />
1.77<br />
Norhydrocodone<br />
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0<br />
6.0e4<br />
4.0e4<br />
2.0e4<br />
0.0<br />
1.36<br />
0.97<br />
0.51 1.71<br />
1.79<br />
1.85 2.15 3.213.36 3.97<br />
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0<br />
26
Selectivity (α)<br />
Selectivity is a measure <strong>of</strong> the difference in the interactions <strong>of</strong> two compounds<br />
with the stationary phase. Selectivity is a function <strong>of</strong> both the stationary phase<br />
and the mobile phase.<br />
α = k 2 /k 1<br />
27<br />
Selectivity (α)<br />
The choice <strong>of</strong> stationary phase will <strong>of</strong>ten have a dramatic effect on the<br />
selectivity <strong>of</strong> analytes.<br />
O<br />
CH 3<br />
OH<br />
CH 3<br />
H<br />
H<br />
HO<br />
H<br />
H<br />
HO<br />
H<br />
H<br />
Estrone<br />
Estradiol<br />
m A U<br />
1 7 5<br />
C18<br />
Column<br />
1+2<br />
1 5 0<br />
1 2 5<br />
1 0 0<br />
α = 1.0<br />
7 5<br />
5 0<br />
2 5<br />
0<br />
1 2 3 4 5<br />
m<br />
i n<br />
m A U<br />
1 0 0<br />
Phenyl Column 1<br />
8 0<br />
6 0<br />
2<br />
α = 2.3<br />
4 0<br />
2 0<br />
0<br />
28<br />
1 2 3 4 5<br />
m<br />
i n
Selectivity (α)<br />
But mobile phase is also a very powerful tool in modulating selectivity.<br />
Column: Gemini 5 µm C6-Phenyl<br />
150 x 4.6mm<br />
Mobile phase: 20mM KH2PO4, pH 2.5;<br />
% organic as noted<br />
Flow rate: 1.0 mL/min<br />
Detection: UV at 220nm<br />
35% Methanol<br />
1. Saccharin<br />
2. p-Hydroxybenzoic Acid<br />
3. Sorbic Acid<br />
4. Dehydroacetic Acid<br />
5. Methylparaben<br />
20% Acetonitrile<br />
29<br />
Column Efficiency (N)<br />
The amount <strong>of</strong> band (peak) broadening or dispersion that occurs in the column<br />
is measured by calculating the column efficiency (N) expressed as the<br />
number <strong>of</strong> theoretical plates in the column:<br />
tR<br />
W 1/2<br />
• Columns that cause a lot <strong>of</strong> peak<br />
broadening have few theoretical<br />
plates.<br />
• Columns that produce very narrow<br />
peaks (little peak broadening) have<br />
a large number <strong>of</strong> theoretical plates.<br />
W<br />
30<br />
Peak Width (W)
Column Efficiency (N) is a<br />
Function <strong>of</strong> Particle Size<br />
Efficiency <strong>of</strong> a well-packed column will be a function <strong>of</strong> several factors, one <strong>of</strong><br />
which will be particle size. As particle size decreases, efficiency will increase.<br />
In addition, back-pressure also increases as particle size decreases.<br />
10 µm<br />
5 µm<br />
3 µm<br />
Core-Shell<br />
sub-2 µm<br />
50,000 P/m<br />
100,000 P/m<br />
150,000 P/m<br />
300,000 P/m<br />
300,000 P/m<br />
Efficiency<br />
Back-Pressure<br />
31<br />
The Core-Shell Advantage<br />
Fully Porous Particles<br />
Columns packed with core-shell particles will deliver<br />
significantly higher efficiency (N) than columns packed with<br />
fully-porous particles <strong>of</strong> the same diameter.*<br />
Fully Porous Particles<br />
w 1/2<br />
Injected<br />
Sample<br />
Band<br />
* Gritti et al., Journal <strong>of</strong> Chromatography A, 1217 (2010) 1589<br />
t R<br />
6
The Core-Shell Advantage<br />
Core-Shell Particles<br />
Columns packed with core-shell particles will deliver<br />
significantly higher efficiency (N) than columns packed with<br />
fully-porous particles <strong>of</strong> the same diameter.*<br />
Kinetex Core-Shell Particles<br />
w 1/2<br />
5 6<br />
t R<br />
* Gritti et al., Journal <strong>of</strong> Chromatography A, 1217 (2010) 1589<br />
The Core-Shell Advantage<br />
Comparison<br />
Columns packed with core-shell particles will deliver<br />
significantly higher efficiency (N) than columns packed with<br />
fully-porous particles <strong>of</strong> the same diameter.*<br />
Fully Porous 3 µm C18; 150 x 4.6 mm<br />
N = 166,500 p/m<br />
Kinetex 2.6 µm C18; 150 x 4.6 mm<br />
N = 295,340 p/m<br />
78% Increase<br />
in Efficiency!<br />
* Gritti et al., Journal <strong>of</strong> Chromatography A, 1217 (2010) 1589<br />
34
The Core-Shell Advantage<br />
Efficiency vs. Diameter<br />
Columns packed with core-shell particles will deliver<br />
significantly higher efficiency (N) than columns packed with<br />
fully-porous particles <strong>of</strong> the same diameter.*<br />
450<br />
Efficiency( p/m)<br />
400<br />
350<br />
300<br />
250<br />
200<br />
150<br />
100<br />
50<br />
Fully Porous<br />
Core-Shell<br />
0<br />
5 3.5/3.6 2.5/2.6 1.7 1.3<br />
Particle Diameter (µm)<br />
*Farcas et al., <strong>HPLC</strong> 2012 Conference<br />
The van Deemter Equation<br />
The three principle factors that cause band broadening and a decrease in<br />
column efficiency are described by the van Deemter equation:<br />
e<br />
e<br />
*<br />
Simplified version:<br />
H =<br />
A · d p + B/µ + C · d e<br />
2 · µ<br />
Particle size<br />
Particle size<br />
Linear velocity (flow rate)<br />
Mass Transfer<br />
36<br />
Linear velocity (flow rate)<br />
Mobile phase viscosity
The Core-Shell Advantage<br />
A-term<br />
H =<br />
A · d p + B/u + C · d e<br />
2 · u<br />
Eddy Diffusion<br />
Multi-path Effect<br />
w 1/2<br />
t R<br />
6<br />
The Core-Shell Advantage<br />
B-term<br />
H =<br />
A · d p + B/u + C · d e<br />
2 · u<br />
Longitudinal diffusion<br />
w 1/2<br />
t R<br />
6
The Core-Shell Advantage<br />
Fully Porous C-term<br />
H =<br />
A · d p + B/µ + C · d e<br />
2 · µ<br />
Mass Transfer (Kinetics)<br />
Dispersion due to resistance to mass transfer<br />
A<br />
B<br />
39<br />
The Core-Shell Advantage<br />
Core-Shell C-term<br />
H =<br />
A · d p + B/µ + C · d e<br />
2 · µ<br />
Dispersion due to resistance to mass transfer<br />
Mass Transfer (Kinetics)<br />
A<br />
B
The Core-Shell Advantage<br />
h vs. v Comparison<br />
H =<br />
A · d p + B/µ + C · d e<br />
2 · µ<br />
Reduced plate height h<br />
15<br />
10<br />
5<br />
4.6 x 100 mm Luna-C 18<br />
(2)<br />
Eddy dispersion<br />
Longitudinal diffusion<br />
Solid-liquid mass transfer<br />
Reduced plate height h<br />
15<br />
10<br />
5<br />
4.6 x 100 mm Kinetex-C 18<br />
Eddy dispersion<br />
Longitudinal diffusion<br />
Solid-liquid mass transfer<br />
0<br />
0 5 10 15 20<br />
Reduced velocity ν<br />
0<br />
0 5 10 15 20<br />
Reduced velocity ν<br />
*Gritti and Guiochon, 2012. LC-GC 30(7) 586-595.<br />
Effect <strong>of</strong> Column Length<br />
on Efficiency<br />
For a given particle size, column efficiency will be directly proportional to the<br />
length <strong>of</strong> the column. However, pressure and elution time will also be<br />
directly proportional to the column length.<br />
5cm<br />
10cm<br />
15cm<br />
25cm<br />
5,000 Plates<br />
8 min<br />
50 Bar<br />
10,000 Plates<br />
16 min<br />
100 Bar<br />
15,000 Plates<br />
24 min<br />
150 Bar<br />
25,000 Plates<br />
40 min<br />
250 Bar<br />
42
Balancing Column Length<br />
and Particle Size<br />
Column<br />
Length<br />
(mm)<br />
Efficiency dp<br />
5µm<br />
250 25,000<br />
150 15,000<br />
100 10,000<br />
50 5,000<br />
43<br />
Balancing Column Length<br />
and Particle Size<br />
Column<br />
Length<br />
(mm)<br />
Efficiency dp<br />
5µm<br />
Efficiency dp<br />
3µm<br />
250 25,000 37,500<br />
150 15,000 22,500<br />
100 10,000 15,000<br />
50 5,000 7,500<br />
44
Balancing Column Length<br />
and Particle Size<br />
Column<br />
Length<br />
(mm)<br />
Efficiency dp<br />
5µm<br />
Efficiency dp<br />
3µm<br />
Efficiency<br />
sub-2µm /<br />
Core-shell<br />
250 25,000 37,500<br />
150 15,000 22,500 45,000<br />
100 10,000 15,000 30,000<br />
50 5,000 7,500 15,000<br />
45<br />
Balancing Column Length<br />
and Particle Size<br />
Column<br />
Length<br />
(mm)<br />
Efficiency dp<br />
5µm<br />
Efficiency dp<br />
3µm<br />
Efficiency<br />
sub-2µm /<br />
Core-shell<br />
%Reduction in<br />
Analysis Time<br />
250 25,000 37,500<br />
150 15,000 22,500 45,000 33<br />
100 10,000 15,000 30,000 60<br />
50 5,000 7,500 15,000 80<br />
46<br />
Use shorter columns packed with smaller particles<br />
to reduce analysis time while maintaining/improving<br />
efficiency!!
Effect <strong>of</strong> Flow Rate on<br />
Efficiency<br />
Effect <strong>of</strong> Flow Rate on Column Efficiency (100x4.6mm)<br />
35000<br />
30000<br />
Core-Shell ~2 mL/min<br />
Core-Shell 2.6<br />
Luna 3u<br />
N (Plates/column)<br />
25000<br />
20000<br />
15000<br />
3µ ~1.5 mL/min<br />
Luna 5u<br />
10000<br />
5000<br />
5µ ~1 mL/min<br />
47<br />
0<br />
0 0.5 1 1.5 2 2.5 3 3.5<br />
Flow rate (ml/min)<br />
Quick Review<br />
The chromatographic measurements that we have discussed so far will all play<br />
a significant role in method development.<br />
1. Retention factor (k’) – retention <strong>of</strong> analyte relative to void t 0<br />
• Controlled by modulating % strong mobile phase<br />
2. Peak asymmetry – peak shape (fronting, tailing, symmetrical)<br />
• Result <strong>of</strong> secondary interactions (e.g. Ionic in RP mode)<br />
• Sensitive to sample loading & sample solvent effects<br />
3. Selectivity (α) – difference in the k’ <strong>of</strong> two analytes<br />
• Will be determined by mobile phase composition and nature <strong>of</strong><br />
stationary phase<br />
4. Efficiency (N) – function <strong>of</strong> peak width and retention<br />
• Determined by particle size, column length, flow rate<br />
• Column packing will affect efficiency (vendor)<br />
48
Any Questions?<br />
49<br />
Resolution: The Goal <strong>of</strong> Chromatography<br />
50
Resolution: The Goal <strong>of</strong><br />
Liquid Chromatography<br />
The goal and the purpose <strong>of</strong><br />
liquid chromatography is to<br />
resolve the individual<br />
components <strong>of</strong> a sample from<br />
each other so that they may be<br />
identified and/or quantitated.<br />
51<br />
Resolution: The Goal <strong>of</strong><br />
Liquid Chromatography<br />
Resolution is a measure <strong>of</strong> how well two peaks are<br />
separated from each other.<br />
It is calculated as the difference in retention time <strong>of</strong> two eluting peaks divided by<br />
the average width <strong>of</strong> the two peaks at the baseline.<br />
R (resolution) = RT B - RT A / .5 (width A + width B )<br />
52
Resolution: The Goal <strong>of</strong><br />
Liquid Chromatography<br />
α<br />
(1) Retention time for the two<br />
peaks will be a function <strong>of</strong><br />
retention factor (k’).<br />
k’<br />
(2) The selectivity (α) will also<br />
affect the retention time<br />
values for the two peaks.<br />
(3) Peak width will be a function<br />
<strong>of</strong> column efficiency (N) and<br />
asymmetry (Asym).<br />
53<br />
N, Asym<br />
Resolution: The Goal <strong>of</strong><br />
Liquid Chromatography<br />
The equation below allows us to calculate the relative importance <strong>of</strong> each <strong>of</strong><br />
these three factors in overall resolution:<br />
Efficiency<br />
Retention factor<br />
Selectivity<br />
It is important to note that you (the analyst) have control over each <strong>of</strong> those<br />
factors through your choice <strong>of</strong> <strong>HPLC</strong> column and running conditions:<br />
1. Efficiency (N)→Particle size/morphology and column length<br />
2. Selectivity (α) → Stationary phase and mobile phase<br />
3. Retention factor (k’) → Stationary phase and mobile phase<br />
54
Resolution: The Relative<br />
Effectiveness <strong>of</strong> k’, α, and N<br />
Most important<br />
determinant <strong>of</strong><br />
resolution!!<br />
Constant increase in<br />
resolution<br />
Ineffective after k’ ~10<br />
55<br />
The Impact <strong>of</strong> Efficiency<br />
on Resolution<br />
Modulating column efficiency is a very effective way to optimize resolution.<br />
There is a strong, linear correlation between N and R s<br />
, but it is not a 1:1 ratio.<br />
Column efficiency is a flexible tool because we can easily modify it via changes<br />
in particle size and column length.<br />
Column<br />
Length<br />
(mm) Efficiency 5µm Efficiency 3µm<br />
Efficiency<br />
sub-2µm /<br />
Core-shell<br />
250 25,000 37,500<br />
56<br />
More Pressure<br />
Longer Run<br />
Time<br />
150 15,000 22,500 45,000<br />
100 10,000 15,000 30,000<br />
50 5,000 7,500 15,000<br />
More efficiency/resolution<br />
More Back-Pressure
The Impact <strong>of</strong> Efficiency<br />
on Resolution<br />
Relative Resolution<br />
2.5<br />
2<br />
1.5<br />
1<br />
0.5<br />
Doubling column<br />
efficiency increases<br />
R s by a factor <strong>of</strong> 1.4x<br />
57<br />
0<br />
0 10000 20000 30000 40000<br />
Column Efficiency (Plates)<br />
mAU<br />
The Impact <strong>of</strong> Efficiency<br />
on Resolution<br />
100<br />
80<br />
60<br />
40<br />
5 µm<br />
80,000 P/m<br />
20<br />
0<br />
0 0.5 1 1.5 2 2.5 3 3.5<br />
min<br />
mAU<br />
140<br />
120<br />
100<br />
80<br />
3 µm<br />
150,000 P/m<br />
60<br />
40<br />
20<br />
0<br />
0 0.5 1 1.5 2 2.5 3 3.5<br />
min<br />
58
Optimizing Efficiency for<br />
Maximum Resolution<br />
1. For method development, start with an intermediate column length, packed<br />
with the smallest particle size that system pressure limitations will allow.<br />
• Conventional <strong>HPLC</strong> → 3µm 150x4.6mm or core-shell 100x4.6mm<br />
• UPLC → sub-2µm or core-shell particle<br />
• Work at optimal flow rate for that particle size<br />
2. Fine-tune for maximum productivity:<br />
• Excessive resolution → shorter column, increase flow rate<br />
• Insufficient resolution → longer column; modify flow rate to compensate<br />
for pressure<br />
59<br />
The Impact <strong>of</strong> Retention<br />
Factor on Resolution<br />
Retention factor is the most important, yet limited, factor in determining<br />
resolution. It is crucial to have a reasonable k’ value because analytes must be<br />
retained in order to separate them. The drawback is that at high k’ values,<br />
passive diffusion causes extensive band broadening and loss <strong>of</strong> performance.<br />
60
Optimizing Retention Factor<br />
for Maximum Resolution<br />
1. Adjust k’ value to be between 2 and 10<br />
• In RP, adjust % <strong>of</strong> organic (acetonitrile or methanol)<br />
• Altering nature <strong>of</strong> stationary phase/media can modulate k’ as well<br />
2. At k’ < 2, have sub-optimal resolution<br />
• May also have interference from solvent, non-retained components<br />
3. At k’ values > 10, band broadening due to diffusion limits resolution gain<br />
• In RP, complex mixtures <strong>of</strong> polar and non-polar components will require<br />
gradient for optimal performance/run time balance<br />
• Polar stationary phases can the “total elution window” <strong>of</strong> complex<br />
mixtures in isocratic mode<br />
61<br />
The Impact <strong>of</strong> Selectivity<br />
on Resolution<br />
Small changes in selectivity can have a dramatic effect on retention. This<br />
is one <strong>of</strong> the reason why the same stationary phases from different<br />
manufacturers can sometimes give very different results, and also why<br />
changes to mobile phase composition can alter the results so strongly.<br />
62
<strong>Method</strong> <strong>Development</strong> Exercise 1:<br />
Optimization to Reduce Analysis Time and<br />
Increase Productivity<br />
63<br />
Mupirocin Impurity Pr<strong>of</strong>ile<br />
Column: C8 250 x 4.6mm 5µm<br />
Mobile phase:<br />
Flow rate:<br />
Components:<br />
70 / 30 0.1M Ammonium acetate / THF<br />
1.0 mL/min<br />
1-6 = Impurities A - G<br />
7. Mupirocin<br />
Mupirocin<br />
64
0<br />
mAU<br />
Mupirocin: Original <strong>Method</strong><br />
DAD1 A, Sig=240,10 Ref=<strong>of</strong>f (Z:\1\DATA\MT050211\DJGSK 2011-05-02 15-34-44\MUPIROCIN000001.D)<br />
175<br />
7<br />
150<br />
125<br />
100<br />
75<br />
50<br />
25<br />
5<br />
R s<br />
3/4 = 0.63; k’ = 2.3<br />
1<br />
2 3 6<br />
4<br />
~16 min<br />
min<br />
0 2 4 6 8 10 12 14 16<br />
Column:<br />
Luna 5µ C8(2) 250 x 4.6mm<br />
Mobile phase: 70/30 0.1M Ammonium acetate pH 5.7/THF<br />
Flow rate:<br />
1.0 mL/min<br />
65<br />
Step 1. Adjust k’ for better<br />
Resolution<br />
66<br />
Step 1. Reduce % organic to increase k’:<br />
• Increases R s<br />
• Increases run time
Step 2. Optimize Efficiency<br />
and Length<br />
Column<br />
Length<br />
(mm)<br />
Efficiency dp<br />
5µm<br />
Efficiency dp<br />
3µm<br />
Efficiency<br />
sub-2µm /<br />
Core-shell<br />
%Reduction in<br />
Analysis Time<br />
250 25,000 37,500<br />
150 15,000 22,500 45,000 33<br />
100 10,000 15,000 30,000 60<br />
50 5,000 7,500 15,000 80<br />
67<br />
Step 2. Switch to 150x4.6mm 3 µm media:<br />
• Reduces analysis time<br />
• Maintains efficiency<br />
Step 3. Optimize the Flow<br />
Rate<br />
35000<br />
30000<br />
25000<br />
Core-Shell 2.6<br />
Luna 3u<br />
Luna 5u<br />
N (Plates/column)<br />
20000<br />
15000<br />
10000<br />
5000<br />
68<br />
0<br />
0 0.5 1 1.5 2 2.5 3 3.5<br />
Flow rate (ml/min)<br />
Step 3. Increase flow rate to 1.5 mL/min:<br />
• Optimizes efficiency for 3 µm
mAU<br />
80<br />
VWD1 A, Wavelength=240 nm (JL050211\MUPI0003.D)<br />
Mupirocin: Intermediate<br />
<strong>Method</strong><br />
5<br />
7<br />
60<br />
40<br />
1<br />
k’ = 9; R s<br />
3/4 = 1.6<br />
20<br />
2 3 4<br />
6<br />
20 min<br />
0<br />
0 2 4 6 8 10 12 14 16 18<br />
min<br />
Column: Luna 3µ C8(2) 150 x 4.6mm<br />
Mobile phase: 80/20 0.1M Ammonium acetate pH 5.7/THF<br />
Flow rate: 1.5 mL/min<br />
69<br />
R s increased from 0.63 to 1.6<br />
Run time increased from 16 to 20 minutes<br />
Step 4. Switch to Core-Shell Media<br />
Column<br />
Length<br />
(mm)<br />
Efficiency dp<br />
5µm<br />
Efficiency dp<br />
3µm<br />
Efficiency<br />
sub-2µm /<br />
Core-shell<br />
%Reduction in<br />
Analysis Time<br />
250 25,000 37,500<br />
150 15,000 22,500 45,000 33<br />
100 10,000 15,000 30,000 60<br />
50 5,000 7,500 15,000 80<br />
70<br />
Step 4. Switch to 100x4.6mm Core-Shell media:<br />
• Reduce analysis time<br />
• Increase efficiency
0<br />
mAU<br />
Mupirocin: Final Optimized <strong>Method</strong><br />
mAU<br />
250<br />
VWD1 A, Wavelength=240 nm (JL050211\MUPI0006.D)<br />
5<br />
7<br />
200<br />
150<br />
1<br />
R s<br />
3/4 = 2.3<br />
100<br />
8 min<br />
50<br />
2 3 4<br />
6<br />
0<br />
0 1 2 3 4 5 6 7 8 9<br />
min<br />
Column: Kinetex 2.6µ C8 100 x 4.6mm<br />
Mobile phase: 80/20 0.1M Ammonium acetate pH 5.7:THF<br />
Flow rate: 1.5 mL/min (2mL/min if pressure allows)<br />
R s<br />
increased from 1.6 to 2.3<br />
Run time decreased from 20 to 8 minutes<br />
71<br />
Initial <strong>Method</strong>:<br />
Mupirocin: Final Optimized <strong>Method</strong><br />
D AD 1 A, Sig= 240,1 0 R ef=o ff ( Z:\1 \DAT A\MT 050 211\ DJ G SK 201 1-0 5-0 2 15 -34 -44 \MU PIRO C IN00 0001 .D)<br />
1 75<br />
1 50<br />
1 25<br />
1 00<br />
75<br />
50<br />
25<br />
1<br />
5<br />
R s<br />
3/4 = 0.63<br />
2 3 4<br />
6<br />
7<br />
16 min<br />
0 2 4 6 8 1 0 12 14 16<br />
Final Optimized <strong>Method</strong>:<br />
VWD1 A, Wavelength=240 nm (JL050211\MUPI0006.D)<br />
mAU<br />
250<br />
5<br />
7<br />
200<br />
150<br />
100<br />
1<br />
R s<br />
3/4 = 2.3<br />
8 min<br />
50<br />
2 3 4<br />
6<br />
72<br />
0<br />
0 1 2 3 4 5 6 7 8 9<br />
min<br />
min
Any Questions?<br />
73<br />
Part 1. <strong>General</strong> Chromatographic Theory<br />
Part 2. The Stationary Phase:<br />
An Overview <strong>of</strong> <strong>HPLC</strong> Media<br />
Part 3. Role <strong>of</strong> the Mobile Phase in Selectivity<br />
74
Fully Porous Silica<br />
Polymerization<br />
Alkaline<br />
Tetraethoxysilane<br />
Silica Sol-Gel<br />
99.9% <strong>of</strong> reactive<br />
surface area is<br />
internal<br />
75<br />
Fully Porous Silica<br />
Advantages:<br />
• Ability to derivatize with numerous<br />
bonded phases<br />
• High mechanical strength<br />
• Excellent efficiency<br />
• Highly amenable to modulation <strong>of</strong><br />
material characteristics (pore size,<br />
surface area, etc.)<br />
Disadvantages:<br />
• Dissolution <strong>of</strong> silica at pH > ~7.5 (may<br />
extend with bonded phase)<br />
• Hydrolysis <strong>of</strong> bonded phase at pH
Organosilica Hybrid Particle<br />
Conventional Silica Particle<br />
Organosilica Hybrid Particle<br />
Siloxane<br />
Bridge<br />
Ethane<br />
linkage<br />
Dissolution at pH > 7.5 Stable to pH ~12<br />
77<br />
Organosilica Hybrid Particle<br />
Advantages:<br />
• Extended pH range from 1-12<br />
• Performance and strength <strong>of</strong><br />
conventional silica particle<br />
• Unique selectivity<br />
Disadvantages:<br />
• Fewer stationary phases available<br />
compared to conventional silica (e.g.<br />
cyano, amino)<br />
78
Core-Shell Particle<br />
0.35 µm Porous Shell<br />
2.6 µm Core-Shell<br />
Particle<br />
1.9 µm Solid Core<br />
79<br />
Core-Shell Particle<br />
Advantages:<br />
• 3x the efficiency <strong>of</strong> 5 µm fullyporous<br />
media & 2x the efficiency <strong>of</strong><br />
3 µm media<br />
• Pressures compatible with<br />
conventional <strong>HPLC</strong> systems*<br />
Disadvantages:<br />
• Pressure is still higher than 3 µm<br />
media<br />
• More sensitive to system extracolumn<br />
volumes<br />
• More sensitive to overload in<br />
some cases<br />
80
RP Stationary Phase Classes<br />
Alkyl bonded phases (C18, C8, C4):<br />
Polar-embedded phases:<br />
Fusion<br />
Phenyl phases (Phenyl, PFP):<br />
Polar-endcapped phases:<br />
F<br />
F<br />
F<br />
F<br />
F<br />
Hydro<br />
81<br />
Methylene Selectivity<br />
We use the methylene selectivity test to determine the ability <strong>of</strong> stationary<br />
phase to separate molecules based upon differences in their hydrophobic<br />
character. In general, very hydrophobic bonded phases (e.g. C18) will display<br />
higher levels <strong>of</strong> methylene selectivity than less hydrophobic phases.<br />
m A U<br />
2 5 0<br />
C18<br />
2 0 0<br />
1 5 0<br />
1 0 0<br />
5 0<br />
0<br />
m A U<br />
2 4 6 8 1 0<br />
m i n<br />
4 0 0<br />
Phenyl<br />
3 0 0<br />
2 0 0<br />
1 0 0<br />
0<br />
2 4 6 8 1 0<br />
m i n<br />
82
Methylene Selectivity<br />
0.200<br />
C18 > C8 > C5 ≥ Phenyl > CN > Amino<br />
0.180<br />
0.160<br />
Slope <strong>of</strong> log k vs. # -CH2- units<br />
0.140<br />
0.120<br />
0.100<br />
0.080<br />
0.060<br />
0.040<br />
0.020<br />
0.000<br />
Luna<br />
C18(2)<br />
Synergi<br />
Hydro-RP<br />
Jupiter<br />
C18<br />
Synergi<br />
Max-RP<br />
Luna<br />
C8(2)<br />
Luna C5<br />
Luna<br />
Phe-Hex<br />
Jupiter<br />
C4<br />
Synergi<br />
Polar-RP<br />
Prodigy<br />
Phenyl<br />
Luna<br />
Cyano<br />
Luna<br />
Amino<br />
83<br />
Methylene Selectivity<br />
Columns:<br />
Mobile phase:<br />
Flow rate:<br />
Components:<br />
5µm C18 150x4.6mm<br />
5µm C8 150x4.6mm<br />
5µm Phenyl 150x4.6mm<br />
65:35 Acetonitrile:Water<br />
1 mL/min<br />
Two steroids:<br />
1. Testosterone<br />
2. Methyltestosterone<br />
CH 3<br />
OH<br />
CH 3<br />
H<br />
CH 3<br />
H<br />
CH 3<br />
OH<br />
CH 3<br />
O<br />
H<br />
H<br />
O<br />
H<br />
H<br />
84<br />
Testosterone<br />
Methyltestosterone
Phenyl Selectivity<br />
Columns:<br />
Dimensions:<br />
Mobile phase:<br />
Flow rate:<br />
C18<br />
Phenyl<br />
150 x 4.6 mm<br />
75:25 Methanol:water<br />
1 mL/min<br />
OH<br />
CH 3<br />
O<br />
CH 3<br />
Components:<br />
1. Estrone<br />
2. Estradiol<br />
HO<br />
H<br />
H<br />
H<br />
HO<br />
H<br />
H<br />
H<br />
Estradiol<br />
Estrone<br />
85<br />
Phenyl Selectivity<br />
OH<br />
CH 3<br />
CH 3<br />
O<br />
H<br />
H<br />
C18<br />
m A U<br />
1 7 5<br />
1 5 0<br />
C18<br />
HO<br />
H<br />
Estradiol<br />
H<br />
HO<br />
H H<br />
Estrone<br />
1+2<br />
1 2 5<br />
1 0 0<br />
7 5<br />
5 0<br />
2 5<br />
0<br />
1 2 3 4 5<br />
m in<br />
Phenyl<br />
m A U<br />
1 0 0<br />
Phenyl<br />
1<br />
8 0<br />
2<br />
6 0<br />
4 0<br />
2 0<br />
0<br />
1 2 3 4 5<br />
m in<br />
86
Aqueous Stability <strong>of</strong><br />
Embedded Phases<br />
Nucleic Acid Bases:<br />
Luna C18(2)<br />
Day 1<br />
Polar-Endcapped C18<br />
Day 1<br />
0<br />
2 4 6 8 10 12<br />
Day 2<br />
0<br />
0 2 4 6 8 10 12<br />
Day 6<br />
Phase Collapse!<br />
0<br />
2 4 6 8 10 12 14<br />
min<br />
0<br />
2 4 6 8 10 12 14<br />
87<br />
Aqueous Stability <strong>of</strong><br />
Embedded Phases<br />
LC/MS/MS Analysis <strong>of</strong> ETG & ETS in Urine:<br />
Polar-Endcapped 2.5 µm C18<br />
100x3.0mm<br />
XIC <strong>of</strong> -MRM (6 pairs): 221.200/75.000 Da ID: ETG-1 from Sample 6 (P-2_Hyro-RP_100x4.6_4u_FR600...<br />
1.00e5<br />
9.50e4<br />
9.00e4<br />
8.50e4<br />
Max. 9020.0 cps.<br />
10mM Ammonium formate<br />
8.00e4<br />
7.50e4<br />
7.00e4<br />
6.50e4<br />
ETG<br />
ETS<br />
6.00e4<br />
1. Ethyl glucuronide<br />
2. Ethyl sulfate<br />
Intensity, cps<br />
5.50e4<br />
5.00e4<br />
4.50e4<br />
4.00e4<br />
3.50e4<br />
3.00e4<br />
2.50e4<br />
2.00e4<br />
1.50e4<br />
1.00e4<br />
5000.00<br />
0.00<br />
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5<br />
49 96 144 191 239 287 334 382 429 477 525 572 620<br />
Time, min<br />
XIC <strong>of</strong> -MRM (6 pairs): 221.200/75.000 Da ID: ETG-1 from Sample 6 (P-2_Hyro-RP_100x4.6_4u_FR600...<br />
Max. 9020.0 cps.<br />
1.10e4<br />
ETG<br />
1.00e4<br />
9000.00<br />
ETG<br />
ETS<br />
8000.00<br />
7000.00<br />
Intensity, cps<br />
6000.00<br />
5000.00<br />
4000.00<br />
ETS<br />
3000.00<br />
2000.00<br />
1000.00<br />
0.00<br />
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5<br />
49 96 144 191 239 287 334 382 429 477 525 572 620<br />
Time, min<br />
88
Part 1. <strong>General</strong> Chromatographic Theory<br />
Part 2. Overview <strong>of</strong> <strong>HPLC</strong> Media<br />
Part 3. Role <strong>of</strong> the Mobile Phase in Selectivity<br />
Solvents for RP<br />
Chromatography<br />
Mobile phase selection is much more challenging that stationary phase<br />
selection because the options are limitless. However, in practical method<br />
development, we can dramatically narrow down the options to focus on those<br />
conditions which will give us the highest likelihood <strong>of</strong> success.<br />
Typical RP Solvents:<br />
Weak Solvent:<br />
Water/Buffer<br />
Strong Solvent: Acetonitrile (64)<br />
Methanol (34)<br />
Composite mixtures (1)<br />
THF (1)<br />
Frequency <strong>of</strong> use
Solvent Selectivity<br />
The elution strength <strong>of</strong> a given solvent is determined by its hydrophobicity (e.g.<br />
heptane would be stronger than hexane because it is more hydrophobic). The<br />
selectivity <strong>of</strong> a solvent, however, is determined by its polar characteristics<br />
(e.g. heptane and hexane would have the same solvent selectivity).<br />
Methanol is a strong proton<br />
donor and a strong proton<br />
acceptor in hydrogen bonding.<br />
C H 3<br />
OH<br />
Acetonitrile has a dipole<br />
moment but is only a very weak<br />
proton acceptor in hydrogen<br />
bonding.<br />
N CH 3<br />
δ − δ +<br />
Tetrahyr<strong>of</strong>uran is able to<br />
accept a proton in hydrogen<br />
bonding but cannot donate a<br />
proton.<br />
O<br />
Solvent Selectivity<br />
Optimum Separation <strong>of</strong> 4 Steroids in Different Solvents:
Solvent Screening for<br />
Isocratic <strong>Method</strong>s<br />
1. Start at high % acetonitrile and work backwards until k’ is 2-10 (if possible)<br />
80% ACN<br />
40% ACN<br />
25% ACN<br />
21% ACN<br />
k’ = 0<br />
k’ ~ 0.8<br />
k’ ~ 6<br />
k’ ~ 11<br />
Solvent Screening for<br />
Isocratic <strong>Method</strong>s<br />
2. Repeat with alternative solvent:
Buffer Selection for<br />
RP-<strong>HPLC</strong><br />
= Typical for LC/UV<br />
= Typical for LC/MS<br />
Effect <strong>of</strong> pH on<br />
Base Silica<br />
Any silica-based RP material will have some residual silanols left after<br />
bonding and end-capping. These Si-OH groups can be deprotonated at<br />
values above pH ~3.5. The deprotonated silanols are more likely to engage in<br />
ion-exchange with basic analytes, leading to peak tailing.<br />
pH 3.5<br />
Si<br />
Si<br />
Si<br />
O<br />
O<br />
Si<br />
O<br />
O Si<br />
O<br />
O Si<br />
OH<br />
OH<br />
OH<br />
Si<br />
Si<br />
Si<br />
O<br />
O<br />
Si<br />
O<br />
O Si<br />
O<br />
O Si<br />
O<br />
OH<br />
O<br />
• Silanols protonated<br />
• Less ion-exchange<br />
• Less peak tailing<br />
• Silanols deprotonated<br />
• Increased ion-exchange<br />
• Increased peak tailing
Effect <strong>of</strong> pH on Analyte<br />
Ionization<br />
The primary mechanism <strong>of</strong> retention in RP chromatography is hydrophobic<br />
interaction. Ionizing compounds will cause them to behave as more polar<br />
species, and reduce their hydrophobic interaction with the stationary phase,<br />
leading to decreased retention.<br />
• More hydrophobic<br />
• More strongly retained<br />
• Less hydrophobic<br />
• Less strongly retained<br />
• More hydrophobic<br />
• More strongly retained<br />
• Less hydrophobic<br />
• Less strongly retained<br />
The ionization state <strong>of</strong> a molecule will be determined by the pH <strong>of</strong> the mobile<br />
phase. Therefore, mobile phase pH will dictate retention behavior <strong>of</strong><br />
analytes with ionizable functional groups.<br />
Effect <strong>of</strong> pH on Analyte<br />
Ionization<br />
Acidic Compounds:<br />
Basic Compounds:<br />
Retention Factor (k’)<br />
Retention Factor (k’)
Effect <strong>of</strong> pH on Analyte<br />
Ionization<br />
Alkaline<br />
H +<br />
Acidic<br />
Alkyl Stationary Phase<br />
Aqueous Mobile Phase<br />
Effect <strong>of</strong> pH on Analyte<br />
Ionization<br />
Alkaline<br />
H +<br />
Acidic<br />
H +<br />
Alkyl Stationary Phase<br />
Aqueous Mobile Phase
Effect <strong>of</strong> pH on Analyte<br />
Retention<br />
Amitriptyline (pKa 9.4) = (B)ase Toluene = (N)eutral Naproxen (pKa 4.5) = (A)cid<br />
B<br />
A<br />
A<br />
B<br />
A<br />
B<br />
N<br />
N<br />
N<br />
Gradient Analysis<br />
The gradient slope is analogous to solvent strength in isocratic elution.<br />
Isocratic Solvent Strength:<br />
Increasing the solvent strength<br />
reduces analysis time but also<br />
reduces resolution.<br />
Decreasing the solvent strength<br />
increases resolution at the cost<br />
<strong>of</strong> increased analysis time.<br />
Solvent strength sometimes<br />
affects selectivity<br />
Gradient Slope:<br />
Increasing the gradient slope<br />
reduces analysis time but also<br />
reduces resolution.<br />
Decreasing the gradient slope<br />
increases the resolution at the<br />
cost <strong>of</strong> increased analysis time.<br />
Gradient slope sometimes<br />
affects selectivity<br />
The goal <strong>of</strong> gradient elution is to optimize resolution while<br />
minimizing analysis time.
Temperature in<br />
<strong>HPLC</strong> <strong>Method</strong>s<br />
The use <strong>of</strong> temperature in <strong>HPLC</strong> method development presents a challenge<br />
because it can have unpredictable effects on selectivity.<br />
The use <strong>of</strong> elevated temperatures will:<br />
1. Reduce mobile phase viscosity and back-pressure. This can allow you to<br />
operate at higher flow rates, or to use longer columns/smaller particle sizes.<br />
2. Reduce elution time.<br />
3. Improve method reproducibility (as opposed to operating at room<br />
temperature).<br />
However, it is impossible to determine if the use <strong>of</strong> elevated temperatures will<br />
help or hinder a specific separation. For complex separations, improvements<br />
in one portion <strong>of</strong> the chromatogram are almost always accompanied by<br />
decreases in another part <strong>of</strong> the same chromatogram.<br />
End <strong>of</strong> Section II<br />
Any Questions?<br />
104
End <strong>of</strong> the <strong>HPLC</strong> <strong>Method</strong> <strong>Development</strong> Portion