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MAXIMIZING PEAK CAPACITY-
Peak capacity is affected by: —Gradient duration (tg) —Flow rates (F) —Column length (L) —Particle size (dp)
Recommended starting flow rates, to be adjusted according to the separation.
We see above that for a 32 minute gradient run on a 4.6 x 20 mm ISTM column, a flow rate of 1.1 mL/min is predicted to maximize the peak capacity. But, for a 4 minute gradient, a fast flow rate of 2.6 mL/min is predicted for maximum peak capacity. We can now run experiments to test the theory.
We observe above that a flow rate of 3 mL/min offers the best solution as far as peak capacity, sol-vent consumption and fast separations.
-0.005
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50
tg
w ww w
w
wt
1P g+=
Gradient Duration
Peak Width-0.005
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50
tg
w ww w
w
wt
1P g+=
Gradient Duration
Peak Width
Peak capacity is a measure of the separation power of a gradient on a particular column.
1tt∆cB
∆cB4
tL
Dcd
LtDbda
L
1P
g
0
0M
2p0
Mp
+⋅⋅
⋅⋅⋅
⋅+⋅⋅+⋅
+=
We can now generate a 3-dimensional plot to examine how the gradient time and flow rate effect the peak capacity of a separation.
wt
1P g+=Start with the simple equation for peak capacity
Make a few substitutions
Neue, U. D., Mazzeo, J. R. J. Sep. Sci. 2001, 24, 921-929.Cheng, Y-F., Lu, Z., Neue, U. Rapid Commun. Mass Spectrom. 2001, 15, 141-151.
0.0 0.1 0.1 0.1 0.2 0.3 0.4 0.5 0.7 1.1 1.5 2.1 3.0 4.2 6.0 8.41.00
2.00
4.008.00
16.0032.00
0
50
100
150
Peak
Cap
acity
Flow Rate [mL/min]
Gradient Duration [min]
Long gradients require
slow flow rates
1.1 mL/min
4.6 x 20 mm, 3.5 µm Column
32 minute gradient
0.0 0.1 0.1 0.1 0.2 0.3 0.4 0.5 0.7 1.1 1.5 2.1 3.0 4.2 6.0 8.41.00
2.00
4.008.00
16.0032.00
0
50
100
150
Peak
Cap
acity
Flow Rate [mL/min]
Gradient Duration [min]
4.6 x 20 mm, 3.5 µm Column
Short gradientsrequire FAST
flow rates
2.6 mL/min
4 minute gradient
2 mL/min790 psi
3 mL/min1200 psi
4 mL/min1650 psi
AU
0.000
0.050
0.080
Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
AU
0.000
0.050
0.080
Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
AU
0.000
0.050
0.070
Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
AU
0.000
0.050
0.070
Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
AU
0.000
0.050
Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
AU
0.000
0.050
Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
1 2 34
5
1 2 34
5
1 2 34
5
P = 32
P = 39
P = 41
XTerra® MS C18 4.6 x 20 mm IS™, 3.5 µm
1 mL/min384 psi
AU
0.000
0.090
Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
P = 231 2 3
4
5
AU
0.000
0.090
Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
P = 231 2 3
4
5
0.63 mL/min2.1 x 20 mm1.28 mL/min3.0 x 20 mm2.16 mL/min3.9 x 20 mm3.0 mL/min4.6 x 20 mmFlow RateColumn Dimensions
0.63 mL/min2.1 x 20 mm1.28 mL/min3.0 x 20 mm2.16 mL/min3.9 x 20 mm3.0 mL/min4.6 x 20 mmFlow RateColumn Dimensions
SCALING SEPARATIONS- To successfully scale separations run on long columns to the 20 mm length columns, we use the following equation to scale the gradient:
We developed this separation on a long column:
Compounds: 1. Caffeine 2. Aniline 3. N-Methylaniline 4. 2-Ethylaniline 5. 4-Nitroanisole 6. N-N-Dimethylaniline Conditions: Column: XTerra® MS C18, 4.6 x 150 mm, 5 µm Mobile Phase A: Water Mobile Phase B: Acetonitrile Mobile Phase C: 100 mM NH4HCO3, pH 10 Flow Rate: 1.4 mL/min Gradient: Time Profile (min) %A %B %C 0.0 80 10 10 20.0 50 40 10 21.0 80 10 10 25.0 80 10 10 Injection Volume: 10 µL Sample concentration: 20 µg/mL Temperature: 30°C Detection: UV @ 254 nm Sampling Rate: 5 pts/sec Filter: 0 (no filter) Instrument: Alliance® 2695 with 996 PDA
To scale the separation to a 20 mm column, we first scale the gradient using the previous equation: We ran a 2.7 minute gradient on a 4.6 x 20 mm IS™, 3.5 µm column at 3 mL/min for a total cycle time of 4 minutes – down from 25 minutes! We further optimized the separation for a total cycle time of 3 minutes.
LC/MS SEPARATIONS-
To scale a gradient
2g1g1
2 ttLL
=×L1 = Long column lengthL2 = Short column length
tg1 = Gradient time on long columntg2 = Gradient time on short column
2
4
3
56
1
AU
0.00
0.02
0.04
0.06
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
AU
0.00
0.02
0.04
0.06
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
Six peaks resolved in a cycle time of 25 minutes.
Can we go faster?
min7.2min2015020 =×
mmmm
AU
0.00
0.02
0.04
0.06
0.08
0.10
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60
AU
0.00
0.02
0.04
0.06
0.08
0.10
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60
24
3
56
1 2 minute elution!
0.00 0.20 0.40 0.60 0.80 1.00Time0
100
%
0
100
%
0
100
%
0
100
%
1: Scan ES+ TIC
1.15e90.88
0.65
0.59
1: Scan ES+ 263
4.05e70.590.65
0.88
1: Scan ES+ 262
1.19e80.65
1: Scan ES+ 233
1.28e80.89
0.00 0.20 0.40 0.60 0.80 1.00Time0
100
%
0
100
%
0.00 0.20 0.40 0.60 0.80 1.00Time0
100
%
0
100
%
0
100
%
0
100
%
1: Scan ES+ TIC
1.15e9
0
100
%
0
100
%
1: Scan ES+ TIC
1.15e90.88
0.65
0.59
1: Scan ES+ 263
4.05e70.590.65
0.88
1: Scan ES+ 262
1.19e8
0.88
0.65
0.59
1: Scan ES+ 263
4.05e70.590.65
0.88
1: Scan ES+ 262
1.19e80.65
1: Scan ES+ 233
1.28e8
0.65
1: Scan ES+ 233
1.28e80.89
Analytes MWCinoxacin 262.2Oxolinic Acid 261.2Nalidixic Acid 232.2
ConditionsColumn: AtlantisTM dC18, 2.1x 20 mm IS™, 3 µmMobile Phase A: H2OMobile Phase B: MeOHMobile Phase C: 1% HCOOH in H2OFlow Rate: 0.4 mL/minGradient: Time Profile
(min) %A %B %C 0.0 50 40 101.0 30 60 10
Injection Volume: 2 µLSample Concentration: 10 µg/mLTemperature: 30 oCInstruments: Alliance® 2795 and Waters ZQTM
Cinoxacin
Nalidixic acid
Oxolinic acid
With a 2.1 mm i.d. column,lower flow rates enable DIRECT flow into the MS and the separation is only 1 minute long!
No flow splitting!
1 Minute!
TIC
Second peak is isotope from oxolinic acid
COST SAVINGS CALCULATIONS- Assume that an HPLC is running about 67% of the year, or 4000 hr. We used the previous separation as an example. We can calculate the solvent cost savings. Assume that 5000 samples need to be ana-lyzed for a study.
4.6 x 150 mm 4.6 x 20 mm IS™
Cycle time (min) 25 3
# of Samples Run per Year
9600 80,000
This is 8.3x as many samples run in one year, on ONE HPLC by using an intelligently designed column!
4.6 x 150 mm 4.6 x 20 mm IS™
Cycle time (min) 25 3
Total time for 5000 samples (hours)
2083 (87 days) 250 (10 days)
Flow rate (mL/min) 1.4 3
Total solvent consumption (L) 175 45
Amount ACN Consumed (L)(~ 25% is ACN)
43.75 11.25
Cost for ACN ($42.50/L) $1860 $480
Cost for waste disposal (~$2.50/L)
$438 $113
Total solvent costs $2298 $593
Cost savings of $1705 in solvents alone. *Additional cost savings in overhead.
To scale a flow rate for different internal diameters
( )( ) 212
1
22 FFx
dd
=
d1 = Diameter of original columnd2 = Diameter of second columnF1 = Flow rate on original columnF2 = Flow rate on second column
Scale from a 4.6 mm i.d. to a 2.1 mm i.d. column.
Flow rate scales from 3 mL/min to 0.6 mL/min.
$23$438Cost for waste disposal (~$2.50/L)
$119$2298Total solvent costs
2.2543.75Amount ACN Consumed (L)(~ 25% is ACN)
$96$1860Cost for ACN ($42.50/L)
0.61.4Flow rate (mL/min)
9175Total solvent consumption (L)
2.1 x 20 mm IS™4.6 x 150 mm
$23$438Cost for waste disposal (~$2.50/L)
$119$2298Total solvent costs
2.2543.75Amount ACN Consumed (L)(~ 25% is ACN)
$96$1860Cost for ACN ($42.50/L)
0.61.4Flow rate (mL/min)
9175Total solvent consumption (L)
2.1 x 20 mm IS™4.6 x 150 mm
Cost savings of $2179!
2.1 x 20 mm IS™, 3.5 µm
AU
0.00
0.02
0.04
0.06
0.08
0.10
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
1
23
4 5 6
2.1 x 20 mm IS™, 3.5 µm
AU
0.00
0.02
0.04
0.06
0.08
0.10
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
AU
0.00
0.02
0.04
0.06
0.08
0.10
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
1
23
4 5 6
For even further cost savings, we can scale the inner diameter from a 4.6 to a 2.1 mm and calculate the savings:
COMPARISON TO MONOLITH- We ran a separation originally run on the monolith, modified the mobile phase, and ran it on the ISTM column. We achieved a better separation.
ISOCRATIC SEPARATIONS-
ConditionsColumn: ChromolithTM SpeedROD RP-18e, 4.6 x 50 mmMobile Phase A: 0.1% TFAMobile Phase B: AcetonitrileFlow Rate: 6.0 mL/minGradient: Time Profile
(min) %A %B0 95 50.4 95 51.2 70 302.2 70 30
Injection Volume: 10.0 µLTemperature: AmbientDetection: UV @ 270 nm
1. Sulfadiazine2. Sulfathiazole3. Sulfamerazine4. Sulfadimidine5. Sulfisoxazole6. Sulfamethoxazole7. Sulfadimethoxine
http://www.merck.de/english/services/chromatographie/hplc/chromolith/pdf/appl_17.pdf
ConditionsColumns: AtlantisTM dC18, 4.6 x 20 mm IS, 3.0 µmMobile Phase A: 0.1% HCOOH in WaterMobile Phase B: 0.1% HCOOH in MeOHFlow Rate: 3.0 mL /minGradient: Time Profile
(min) %A %B0 100 0 4 50 50
Injection Volume: 10.0 µLSample concentration: 20 µg/mLTemperature: 30 °CDetection: UV @ 270 nmInstrument: Alliance® 2795 with 996 PDA
t0 = 0.17 minMax psi = 2240
AU
0.00
0.05
0.10
0.15
Minutes
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
4
5
1
2 36
7A
U
0.00
0.05
0.10
0.15
Minutes
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
AU
0.00
0.05
0.10
0.15
Minutes
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
4
5
1
2 36
7
Half the flow rate Much better peak shapeUnder a 3 minute separation
2.7 Minutes
8.1 mL
2 Minutes
12 mL
12
43
AU
0.00
0.02
0.04
0.06
Minutes
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
12
43
AU
0.00
0.02
0.04
0.06
Minutes
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
AU
0.00
0.02
0.04
0.06
Minutes
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
Under 3 minutes!
Isocratic method development is possible on short columns.
Conditions
Column: SymmetryShieldTM RP18, 4.6 x 20mm IS™, 3.5 µm Mobile Phase A: 0.1% HCOOH in Water Mobile Phase B: 0.1% HCOOH in ACN Flow Rate: 3.0 mL/min Isocratic Conditions: 65% A, 35% B Injection Volume: 10 µL Sample Concentration: 20 µg/mL Temperature: 30 °C Detection: UV @ 220 nm Sampling rate: 10 pts/sec Filter: 0 Instrument: Alliance® 2695 with 996 PDA
1. Ketoprofen 2. Fenoprofen 3. Flurbiprofen 4. Ibuprofen
COLUMN LIFETIME-
CONCLUSIONS – Shorter analysis times can be achieved with shorter particulate columns with excellent results: • Significant cost savings • Good column lifetimes New 20 mm length Intelligent Speed (IS™) columns are available: • Scale-down longer methods • Perform Method Development • Simplify LC/MS Waters can assist with a complete solution: • IS™ columns • Choose from a wide range of chemistries and
configurations • Method development technical support • Waters Instrumentation and Service
Waters, Alliance, Symmetry, SymmetryShield, Atlantis and XTerra are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. ©2003 Waters Corporation
Injection 1Maximum Backpressure = 2500 psi
Injection 1000Maximum Backpressure = 2560 psi
1000 injections on a 4.6 x 20 mm IS™, 2.5 µm particlecolumn with only a negligible increase in backpressure!
Retention time shift = less than 6% difference
Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
1 23
Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
1 23
Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
12 3
Excellent Peak Shape – 1 Minute Elution
1. Atenolol2. Pindolol3. Metoprolol
* = Peak difference due to different lot of plasma
*
*
Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
1
2 3
Injection 1Maximum Backpressure = 2360 psi
Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
1
2 3 Injection 1000Maximum Backpressure = 2410 psi
1000 injections on a 2.1 x 20 mm IS™, 2.5 µm particle column with only a negligible increase in backpressure!
Retention time shift = less than 7% difference
Excellent Peak Shape - 1 Minute Elution
*
* = Peak due to new lot of plasma
1. Atenolol2. Pindolol3. Metoprolol