This presentation describes the benefits of UPLC technology in the analytical laboratory.

1. The Benefits of Using UPLC® in the
Analytical Laboratory
Chromatography Forum of Delaware Valley Poster Session
Jim Alexander
Rohm and Haas Co.
April 17, 2008
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2. Terminology
HPLC – High Performance Liquid Chromatography
Early 1970’s
10, 5, 3.5 µm particles
4.6 mm x 250 mm, 2.1 x 150 mm, and 1.0 x 150 mm
1,000 – 3,000 psi (system max 6,000 psi)
Gradient analysis ~45 min.
UPLC® – Ultra Performance Liquid Chromatography
2004 (Waters Corp.)
1.7 µm particles
2.1 mm x 150 mm, 2.1 x 50 mm, and 1.0 x 50 mm
2,000 – 14,000 psi (maximum pressure 15,000 psi)
Gradient analysis ~4.5 min. Separation
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3. UPLC/UV/MS Instrument
Waters
ACQUITY UPLC
Waters ZMD
UV Detector
Autosampler
UPLC Pump
Column Compartment Mass Spectrometer Separation
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4. The Advantages of UPLC
Speed
Resolution Key advantages
Sensitivity
Instrument Performance
Injection repeatability
Gradient repeatability
Additional advantages
Instrument stability
Column-to-column repeatability
Instrument versatility
Solvent reduction and waste minimization
Separation
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5. Efficiency, Resolution, and Pressure
N = L / h dp N1/2 (α−1) k2
Rs =
α k2+1
4
N = efficiency
Rs = resolution
L = column length
α = selectivity factor
k2 = capacity factor
h = reduced height
equivalent to a theoretical
plate
φuηL
dp = mean particle diameter P=
dp2
P = pressure drop
u = linear velocity
The goal is to obtain separation η = mobile phase viscosity
φ = flow resistance factor
conditions with the highest
resolving power (Rs) possible. This
can be achieved by increasing
efficiency (N).
Separation
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6. The Advantages of sub-2 µm Particles
Speed
5 µm dp 1.7 µm dp, then 9x speed
3.5 µm dp 1.7 µm dp, then 4x speed
Fopt ∝ L/dp
5 µm dp 1.7 µm dp, then 3x flow rate and 3x N
Therefore, L 3x and maintain Rs in 1/9 the time.
Must consider pressure drop
Popt ∝ 1/dp3
5 µm dp 1.7 µm dp, then 25x P
If, L 3x, then 8x P
Separation
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7. The Advantages of sub-2 µm Particles
Resolution
Rs ∝ N N ∝ 1/dp Rs ∝ 1/ dp
5 µm dp 1.7 µm dp, then 3x N and 1.7x Rs
3.5 µm dp 1.7 µm dp, then 2x N and 1.4x Rs
What is the effect of
Rs=0.4
0.5 0.6
1.7x or 70% more
0.7
resolution?
70%
1.25
1.0
0.8
Separation
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7 Waters UPLC Presentation, ASMS, 2006

8. The Advantages of sub-2 µm Particles
Resolution
Rs ∝ N N ∝ 1/dp Rs ∝ 1/ dp
5 µm dp 1.7 µm dp, then 3x N and 1.7x Rs
3.5 µm dp 1.7 µm dp, then 2x N and 1.4x Rs
Sensitivity
w ∝ 1/ N
5 µm dp 1.7 µm dp, then 1.7x S/N
3.5 µm dp 1.7 µm dp, then 1.4x S/N
Separation
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9. The Advantages of sub-2 µm Particles
Speed
5 µm dp 1.7 µm dp, then 9x speed
3.5 µm dp 1.7 µm dp, then 4x speed
Fopt ∝ L/dp
5 µm dp 1.7 µm dp, then 3x flow rate and 3x N
Therefore, L 3x and maintain Rs in 1/9 the time.
Must consider pressure drop
Popt ∝ 1/dp3
5 µm dp 1.7 µm dp, then 25x P
If, L 3x, then 8x P
Separation
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10. The Advantages of UPLC
Separation
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Waters UPLC Presentation, ASMS, 2006
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11. Analysis Conditions as a Function of Particle Size
At constant column length.
optimal flow rate,
d p, µm analysis time, min L, cm
N P, psi
mL/min
5.0 30 25,000 270 1.00 25
3.0 18 42,000 1,300 1.67 25
1.5 9 83,000 10,000 3.33 25
1.0 6 125,000 34,000 5.00 25
J. E. MacNair, K. D. Patel and J. W. Jorgenson, Anal. Chem. 1999, 71, 700.
At constant efficiency.
optimal flow rate,
d p, µm analysis time, min L, cm
N P, psi S/N
mL/min
5.0 30 25,000 270 1.00 25 1
3.0 11 25,000 780 1.67 15 1.7x
1.5 2.7 25,000 3,000 3.33 8 3.3x
1.0 1.2 25,000 6,800 5.00 5 5.0x
Separation
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12. Analysis Conditions using Common Column Formats
Vary particle size and column length. Column i.d. = 2.1 mm
optimal flow rate,
d p, µm analysis time, min L, cm
N P, psi N/min
mL/min
5.0 30 25,000 1,000 0.21 25 800
5.0 18 15,000 600 0.21 15 800
3.5 13 21,000 1,700 0.30 15 1,700
3.5 8.4 14,000 1,200 0.30 10 1,700
3.5 4.2 7,000 600 0.30 5 1,700
2.5 9.0 30,000 4,800 0.42 15 3,300
2.5 6.0 20,000 3,200 0.42 10 3,300
2.5 3.0 10,000 1,600 0.42 5 3,300
1.7 6.1 44,000 15,200 0.62 15 7,200
1.7 4.1 29,000 10,200 0.62 10 7,200
1.7 2.0 15,000 5,100 0.62 5 7,200
Separation
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13. PAH Gradient Separation with 5 µm Column
042307010 2: Diode Array
2.46 Range: 1.642
3.60
1.89
Waters XBridge C18, 5.0 µm
1.4
2.1 x 50 mm 3.17
F = 0.21 mL/min
1.3
2.36
H2O/ACN Gradient
1.2
40C, 1 µL inj.
254 nm 1.73
1.1
2.21
1.0
9.0e-1
8.0e-1
AU
4.23
7.0e-1 3.79
2.70
6.0e-1
5.0e-1
4.0e-1
3.96
3.0e-1
2.83
2.0e-1
5.0 min
1.0e-1
0.63
0.0
Time
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
Separation
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14. PAH Gradient Separation with 1.7 µm Column
Improved Resolution
042307018 2: Diode Array
3.56 Range: 2.028
1.87
1.8
Waters BEH C18, 1.7 µm 2.43
1.7
2.1 x 50 mm
F = 0.21 mL/min
1.6 2.33
H2O/ACN Gradient
1.5
40C, 1 µL inj.
1.4
254 nm 3.13
1.70
2.19
1.3
1.2
1.1
1.0
AU
3.73
9.0e-1 4.16
2.67
4.21
8.0e-1
Narrower peaks
7.0e-1
6.0e-1
5.0e-1
3.92
4.0e-1
2.80
3.0e-1
5.0 min
2.0e-1
1.0e-1 2.98
0.61
0.0
Time
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
Separation
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15. PAH Gradient Separation with 1.7 µm Column
Improved Resolution and 3x Decrease in Analysis Time
042307021 2: Diode Array
1.25 Range: 1.998
0.67
1.8
Waters BEH C18, 1.7 µm 0.86
1.7
2.1 x 50 mm
F = 0.61 mL/min
1.6
0.83
H2O/ACN Gradient
1.5
40C, 1 µL inj. 1.10
1.4
254 nm
0.61 0.78
1.3
1.2
1.1
1.0
1.30
AU
1.44
9.0e-1
1.46
0.95
8.0e-1
7.0e-1
6.0e-1
5.0e-1
1.36
4.0e-1
0.99
3.0e-1
1.7 min
2.0e-1
1.0e-1 1.05
0.25
0.0
Time
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 1.60 1.70
Separation
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16. Comparison of 5.0 µm and 1.7 µm Columns
Increased Sensitivity
041907058 2: Diode Array
1.67 Range: 1.195e-1
2.3x Increase in Signal
1.1e-1
Waters BEH C18, 1.7 µm
Waters XBridge C18, 5.0 µm
2.1 x 50 mm 1.0e-1
Sensitivity
F = 0.21 mL/min
H2O/ACN (40:60)
w ∝ 1/ N
9.0e-2
40C, 1 µL inj.
254 nm 5 µm dp 1.7 µm dp, then 1.7x S/N
Analyte: Toluene 8.0e-2
3.5 µm dp 1.7 µm dp, then 1.4x S/N
7.0e-2
AU
6.0e-2
1.7 µm
5.0e-2
4.0e-2
3.0e-2
5.0 µm
2.0e-2
1.0e-2
Separation
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0.0 Time
1.20 1.40 1.60 1.80 2.00
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17. RP Test Mixture – Isocratic Separation
Peak Area and Retention Time Repeatability
RP Test mix D4, 60:40
041907043 2: Diode Array
Range: 3.196e-1
0.40
0.31
Retention Time
3.0e-1
RP Test Mixture %RSD 0.14% (0.03 s)
Waters BEH C18, 1.7 µm
2.8e-1
Peak Area
2.1 x 50 mm
2.6e-1
%RSD 0.14%
F = 0.60 mL/min
2.4e-1
H2O/ACN (40:60)
40C, 1 µL inj. (partial loop)
2.2e-1
254 nm
Retention Time
2.0e-1
%RSD 0.14% (0.05 s)
1.8e-1
Peak Area
Overlay of 7 injections
N,N-Diethyl-m-Toluamide
1.6e-1
%RSD 0.13%
AU
1.4e-1 0.59
1.2e-1
1.0e-1 0.24
8.0e-2
Toluene
Phenol
6.0e-2
4.0e-2
0.28
2.0e-2
0.0
Time
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70
Separation
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18. Comparison of Conventional and UPLC Separations
070506007 2: Diode Array
2.31 Range: 1.424e-1
5.0e-2
XTerra C18 Column
4.0e-2
1 x 150 mm, 3.5 µm, 50 µL/min
3.0e-2
1.73
2.0e-2
AU
1.0e-2
26.05
22.95
2.96 3.27
0.0
-1.0e-2
2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50
070706009 2: Diode Array
0.37 Range: 5.223e-1
1.6e-1
1.4e-1
1.2e-1
1.0e-1
9-fold reduction UPLC BEH C18 Column
8.0e-2
AU
in analysis time
3.18
1 x 50 mm, 1.7 µm, 150 µL/min
6.0e-2
4.0e-2
2.0e-2
0.64
1.88
0.0
-2.0e-2
Time
2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50
Separation
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19. Comparison of Conventional and UPLC Separations
L AS , 1 0.4 pp m
070506007 2: D iode A rray
R ange: 1.141e-2
-7.0e-3
XTerra C18 Column
-8.0e-3
-9.0e-3
1 x 150 mm, 3.5 µm, 50 µL/min
-1.0e-2
-1.1e-2
-1.2e-2
AU
-1.3e-2
13.2 s
-1.4e-2
peak width
-1.5e-2
-1.6e-2
-1.7e-2
-1.8e-2 T im e
6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00
L AS , 1 0.4 pp m
070706009 2: D iode A rray
1.88 R ange: 2.86e-2
4.0e-3
2.0e-3
1.9 7
-1.709e-9
UPLC BEH C18 Column
-2.0e-3
-4.0e-3
1 x 50 mm, 1.7 µm, 150 µL/min
2.4 s
-6.0e-3
-8.0e-3
peak width
AU
-1.0e-2
-1.2e-2
-1.4e-2
-1.6e-2
-1.8e-2
-2.0e-2
Separation
-2.2e-2
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T im e
1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50
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20. The Benefits
Speed
Methods are developed faster
Samples are analyzed faster, information returned to
clients faster
For example, in 8 h we can run 96 analyses (5 min
method) versus 14 analyses (35 min method)
Quality of results
Data is rugged and reliable
Additional experiments can be run to verify results
without a significant loss of time
Analyst
Approach to solving problems changes
Chances for success improved Separation
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21. The Benefits
Versatility
The ACQUITY UPLC can be used with analytical,
narrow bore, and microbore columns from 10
µL/min to 2 mL/min
Column Chemistry
UPLC BEH C18 1.7 µm columns have been
excellent direct replacements for other HPLC C18
columns
The 1 x 50 mm and 2 x 50 mm formats have been
the most useful for fast separations
Batch to batch differences are minimal
Separation
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