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Page 1
06/05/2011
Rs = N 4
LC Factors affecting
( 1)
k (k+1)
Column length, particle size Stationary phase chemistry, mobile phase chemistry, temperature
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Typical Method Development Parameters - Effects of Selectivity, Efficiency, and Retention on Resolution
Selectivity Impacts Resolution Most
7.00
6.00
5.00
Resolution
4.00
3.00
2.00
1.00
Rs = N/4 ( -1)/
0.00
k/(k+1)
13 14 15
Plates: Alpha: k:
10
11
12
16
17
18
19
20
21
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06/05/2011
7 4 6
pH 4.8 NH4OAc/MeCN
1 2 7
0 2 4
6 5
8
Selectivity and resolution can change with pH Eclipse Plus can be used with many mobile phases and pHs
4
6
min
pH 7 NaPO4/MeCN
7 3 4 5
Conditions: Column: Eclipse Plus C18 4.6 x 100mm, 5um Gradient: 10 90% in 10 minutes Detection: UV 254 nm
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Start at Low pH, Adjust Organic Eclipse Plus C18 Cardiac Drugs with Acetonitrile
mAU
400 300 200 100 0
Column: ZORBAX RRHT Eclipse Plus C18, 4.6 x 50 mm, 1.8 m Mobile Phase: A: 25 mM NaH2PO4 , pH 3.0 B: ACN Flow Rate: 2.0 mL/min Temperature: 30C Detection: UV 240 nm Sample: Cardiac Drugs 1.Pindolol 2. Diisopyridamide 3.Propranolol 4.Dipyridamole 5. Diltiazem
40% ACN
2
RRHT Eclipse Plus C18 allows for very fast optimization of % organic in mobile phase.
Good resolution Fast analysis No time wasted
30% ACN
2
mAU 60 40 20 0 -20 2 4 6 8
20% ACN
To get this k on a 25cm column at 2 mL/min would require 1.5 hours run time!!
k= 44!!
min
10
12
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06/05/2011
Start at Low pH on Eclipse Plus C18 RRHT Cardiac Drugs with Methanol, Changing Organic Modifier
mAU 400 300 200 100 0
Column: ZORBAX RRHT Eclipse Plus C18, 4.6 x 50 mm, 1.8 m Mobile Phase: A: 25 mM NaH2PO4 , pH 3.0 B: MeOH Flow Rate: 2.0 mL/min Temperature: 30C Detection: UV 240 nm Sample: Cardiac Drugs 1.Pindolol 2. Diisopyridamide 3.Propranolol 4.Diltiazem 5. Dipyridamole
70% MeOH
2
50% MeOH
Adjusting MeOH influences retention like adjusting ACN Solvents can be compared quickly with RRHT columns MeOH changes selectivity and gives longer analysis time for these analytes
40% MeOH
8 10
k= 49!!
12 min
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06/05/2011
2
3 4
Bonus-RP
7
Alkyl-C8
7
Si NH O O
Page 8
06/05/2011
Column Efficiency
Number of theoretical plates, N Theoretical plates per metre, N/m Height equivalent to a theoretical plate:
Hmm = Lmm/N
NB: the smaller H, the more efficient the column
Page 9 06/05/2011
small particle
H min
linear flow u
06/05/2011
0.0025
Column: ZORBAX Eclipse XDB-C18 Dimensions: 4.6 x 50/30mm Eluent: 85:15 ACN:Water Flow Rates: 0.05 5.0 mL/min Temp: 20C Sample: 1.0 L Octanophenone in Eluent
H = A + B/u + Cu
5um
HETP (cm)
0.0020
0.0015
3.5um
0.0010
0.0005
1.8um
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
Page 11 06/05/2011
Resolution
Shorter Columns with Smaller Particles Plates Selectivity Retention
Rs =
N
-1
4
L
k' k'+1
Column Length = N
dp
dp/2
Particle Size
Particle Size
=
=
N
P
000990P2.PPT
06/05/2011
Analysis Time*
-33% -50%
Pressure
-67% -80%
-90%
* Reduction in analysis time compared to 150 mm column; all columns 4.6-mm i.d.
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06/05/2011
HPLC, 40C
10
min
RRHT SB-C18 2.1mm x 50mm 1.8m 2.40ml/min, 95C Analysis Time: 0.4min
RRLC, 95C
27x faster
0 0.2 0.4 min 0.6
>20X faster
PW = 197msec
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06/05/2011
Tools
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06/05/2011
There is a trade-off between speed and resolution, but it is often possible to have a mixture of slightly faster analysis with more resolution
This comes at a price :Much higher system backpressure
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06/05/2011
Pressure Equation
viscosity flow length
FL P = 0 2 2 K r dp
specific permeability column radius particle diameter
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The particle has a solid core (1.7um) and porous outer layer with a 0.5um diffusion path
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SEM of Cores
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Comparison of Particle Size Distributions Between Totally Porous and Poroshell 120 Particles
Very narrow particle size distribution reduces the A term of the van Deemter equation (Eddy diffusion)
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06/05/2011
Coacervation is the process by which small sol particles come together to form the porous particle
Like the finely controlled pore size/particle size process of ZORBAX 1 step reduces variability more reproducible
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06/05/2011
Comparison of 4.6 x 250 mm 5 um to Poroshell 120 EC-C18 4.6 x 100 mm, 2.7um
mAU 100 80 60 40 20 0 mAU 250
1.719 7.037
16.151 16.435
9.712
11.116
15.248
5
2.311
10
15
20
23.076
110 bar
20.687
25
29.290
Time %B 0 8 33 33 34 33 Column: Eclipse Plus C18 4.6 x 250mm, 5um Flow Rate: 1 mL/min
Mobile Phase: A: 0.1% formic acid in Water B: 0.1% formic acid in ACN
11.596
Time %B 0 8 12 33 13.2 33 Column: 4.6 x 100mm Poroshell 120 EC-C18, 2.7um Flow Rate: 1 mL/min
Sulfadiazine, Sulfathiazole Sulfapyridine Sulfamerazine, Sulfamethazine, Sulfamethazole, Sulfamethoxypyridazine, Sulfachloropyridazine Sulfamethoxazole, Sulfadimethoxine
12.674
30
min
325 bar
2.606 5.450 5.920 4.437 4.558
50
0
Faster analysis (shorter column higher flow rate) More Sensitive (smaller particle sharpens peak) Better Optimized (faster analysis allows better tweaking) Both run on Agilent 1100 G1315B DAD (under 400 bar) No change in sample prep required, 2 micron frit used on both columns
10 15 20 25 30 min
2.189
3.867
Page 23
06/05/2011
Comparison of 4.6 x 250 mm 5 um to Poroshell 120 EC-C18 4.6 x 100 mm, 2.7um
See conditions previous slide
12.674
mAU 100
11.116
11.596
9.712
15.248
60 40 20 0
250 200
325 bar
1.719 2.189
2.311
2.606
4.437 4.558
5.450
150 100
50
0
3.867
5.920
7.037
mAU
10
15
20
23.076
110 bar
20.687
25
29.290
80
16.151 16.435
30
min
min
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Resolution
N k Rs = 4 k 1
Efficiency Retention Selectivity
Page 26
1
L = Length rc = column radius df = film thickness T = temperature
N=
k =
(gas, L, rc)
(T, df, rc)
(T, phase)
06/05/2011
Siloxane polymers
Poly(ethylene) glycols
Porous polymers
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06/05/2011
Optimizing Selectivity
Match analyte polarity to stationary phase polarity -like dissolves like(oil and water dont mix)
Take advantage of unique interactions between analyte and stationary phase functional groups
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06/05/2011
6 Time (min.)
10
12
14
3
Time (min.)
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06/05/2011
Length (m) 15 30 60
0.25 mm ID n/m = 4630 (for k = 5)
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06/05/2011
15 m
30 m
Double the plates, double the time but not double the the resolution
60 m
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06/05/2011
4
3 5
0 0 .5
.5
1 0 .0
1.0
1 0 .5
1.5
2 0 .0
2.0
2 0 .5
2.5
3 0 .0
3.0
3 0 .5
3.5
4 0 .0
4.0
4 0 .5
4.5
5.0
5 0 .0
5 0 .5
5.5
6 0 .0
6.0
6 0 .5
6.5
7 0 .0
7.0
7 0 .5
7.5
8 0 .0
8.0
8 0 .5
8.5
9 0 .0
9.0
1. 2. 3. 4. 5.
4 3
0 0 .2
0 0 .4
1.0 0.60
0 0 .8
1 0 .0
1 0 .2
1 0 .4
2.0 1.60
1 0 .8
2 0 .0
2 0 .2
2 0 .4
3.0 2.60
2 0 .8
3 0 .0
3 0 .2
3 0 .4
4.0 3.60
3 0 .8
4 0 .0
4 0 .2
4 0 .4
5.0
4 0 .6
4 0 .8
5 0 .0
5 0 .2
5 0 .4
6.0
5 0 .6
5 0 .8
BTEX Carrier: Helium, 36 cm/sec at 40c Oven : 40C for 3 min, 5/min to 100C
Page 32 06/05/2011
Length Summary
If you Decrease Length: Efficiency Resolution Analysis Time Pressure Cost Decrease Decrease Decrease Decrease Decrease
Page 33
06/05/2011
Resolution
N k Rs = 4 k 1
Efficiency Retention Selectivity
Page 34
1
L = Length rc = column radius df = film thickness T = temperature
N=
k =
(gas, L, rc)
(T, df, rc)
(T, phase)
06/05/2011
Different Column I. D.
Equal Phase Ratios
Column : DB-624 30 m, 0.53 mm, 3 m Carrier: Helium, 40 (cm/sec) Oven: 65C Injection: Split Detector: FID
Column :
10 Time (min)
15
20
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06/05/2011
PHASE RATIO ( )
Film Thickness
Column Dimensions
Phase Ratio
30 m x .53 mm x 3.0 m
30 m x .32 mm x 1.8 m KC = k = r 2df
44
44
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06/05/2011
0.18
0.20
30-45
25-40
0.25
30 meters Hydrogen pressures x 1/2 0.32 0.45 0.53
Page 37
15-25
10-20 3-7 2-4
06/05/2011
Diameter Summary
Pressure
Capacity Flow rate
Increase
Decrease Decrease
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06/05/2011
N2
1.00
H (min)
He H2
60
u (cm/sec)
Page 40 06/05/2011
Plates/m
k = 5
06/05/2011
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06/05/2011
Spearmint Oil
10.6 min
-16.8 min (0.18 mm, H2 Carrier)
17.7 min
-9.7 min (0.18 mm, He Carrier)
27.4 min
13
18
23
28
Time (min)
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06/05/2011
10.6 min
17.7 min
27.4 min
13
18
23
28
Page 47
06/05/2011
Resolution Maintained
Compound Resolution
0.18 mm Helium 1.59 1.73 35%
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06/05/2011
Questions?
Page 60
06/05/2011