3gradient Handouts

Gradient Operation in HPLC
Dr. Shulamit Levin Medtechnica

levins@medtechnica.co.il levins@medtechnica.co.il http://www.forumsci.co.il/HPLC http://www.forumsci.co.il/HPLC http://shulalevin.tripod.com http://shulalevin.tripod.com
Gradient Operation in HPLC
Introduction - Optimizing Gradient Separations

The following diagram illustrates the cycle time parameters that are used in a typical gradient
Dr. Shulamit Levin Medtechnica

levins@medtechnica.co.il levins@medtechnica.co.il http://www.forumsci.co.il/HPLC http://www.forumsci.co.il/HPLC http://shulalevin.tripod.com http://shulalevin.tripod.com
% Organic
Final Hold
Gr ad ien t
Inject
Gradient Time Total Cycle Time
Re-equilibration
Outline
Introduction Strategies for Higher Throughput Gradient Separations to Achieve Maximum Throughput and Optimal Resolution system solutions method solutions
Typical Problems Encountered in Gradient Chromatography

Non-reproducible retention times Difficulties to transfer from analytical to narrowbore columns Long reequilibration times Long cycle times (injection to injection) More efficient analyses desired
Dr. Shulamit Levin, Medtechnica
Introduction - Options to Improve Sample Throughput

System Solutions:
Reduce Gradient Delay Volume Decrease Re-equilibration time time Reduce Injection Cycle time Modify Instrument Use Multiple Parallel Columns Adjust Detector Sampling Rate
Outline
Introduction Strategies for for Higher Throughput Gradient Separations to Achieve Maximum Throughput and Optimal Resolution
system system solutions reduce gradient delay volume decrease decrease re-equilibration re-equilibration time reduce injection cycle time method solutions
Method Solutions:
Use Shorter Gradients Use Higher Flow Rates Use Shorter Columns Use a Smaller Particle Size Decrease Re-equilibration Time Increase Temperature
Test Probe Structures

S O
0.20
Initial Separation and Conditions

C CH3 O
N N N N
H2C CH
Conditions: Column: Symmetry C18, 5 m, 4.6 X 50 mm Mobile phase: A=0.1% TFA in water, B=0.1% TFA in acetonitrile
0.18 0.16 0.14
OH
NH2
0.12 0.10 AU 0.08
1-hydroxy-7-aza-benzotriazole
H3C
methyl 3-amino-2-thiophenecarboxylate
C
Gradient: 0-60% B in 8 minutes Column temperature: 30.0 C Flow rate: 1 mL/min.
0.06 0.04
O S O NH2
H2N C
0.02 0.00 0.00 0.50 1.00 1.50 2.00 Minutes 2.50 3.00 3.50 4.00
Detector: 254 nm Injection volume: 1 L
4-methylbenzene sulfonamide
4-aminobenzophenone
A - 1-hydroxy-7-aza-benzotriazole B - 4-methylbenzene sulfonamide C - methyl 3-amino-2-thiophenecarboxylate D - 4-aminobenzophenone
H. Weller, Bristol-Myers Squibb Pharmaceutical Research Institute
Volumes in an HPLC System

System Volume
Waters 2690 <650 L
B D
Determination of System Precolumn Volume

Definition: Delay volume is the volume of plumbing between the point the gradient is formed and the inlet of the column.
Measured by linear gradient Measured by step gradient
1.0 0.8 0.6
Column Volume Column Detector

Height
1.0 0.8 0.6
Solvent delivery
Injector
Height
0.4 0.2 0.4 0.2
Proportioning Valve
Extra Column Volume
Volume (L)
Volume (L)
System components affecting dwell volume: Pump Gradient Mixers Injector
Effect of Precolumn Volume

Reducing Delay Volume
in theory
Final Hold
Gradient Shape and Precolumn Volume

Theoretical Gradient No column , 1.00 mL/min 3.9x150 mm, 1.00 mL/min 2.1x150 mm, 0.29 mL/min
real life
Final Hold
% Organic
% Organic
Gr
Inject
Gradient Time
Re-equilibration
Inject
Gr ad ien t
ad ie
nt
%B
Gradient Time
Re-equilibration
Total Cycle Time
Total Cycle Time
Delay Volume
0 5 10
Re-equilibration Time
15 Minutes 20 25 30
Outline
Introduction Strategies for Higher Throughput Gradient Separations to Achieve Maximum Throughput and Optimal Resolution
system solutions reduce gradient delay delay volume decrease re-equilibration time reduce injection cycle time time method solutions
Calculation of of Gradient Equilibration Volume
Re-equilibration Re-equilibration is is a a necessary necessary part part of of gradient gradient chromatography. chromatography. Both Both the the HPLC HPLC system system and and the the column column must must be be at at initial initial conditions conditions at at the the beginning beginning of of each each run run to to ensure ensure reproducible reproducible chromatographic chromatographic separations. separations. The The re-equilibration re-equilibration volume volume can can be be divided divided into into two two parts, parts, the the system system washout washout and and the the column column re-equilibration. re-equilibration. For For good good system/column system/column equilibration equilibration
Gradient Shape and Re-equilibration

Theoretical Gradient No column , 1.00 mL/min 3.9x150mm, 1.00 mL/min 2.1x150mm, 0.29 mL/min
%B
Delay Volume
0 5 10
Re-equilibration Time
15 Minutes 20 25 30
Column Re-equilibration
3.9 x 150 mm 2.1 x 150 mm 1.0 x 150 mm 0 1.0 mL/min 0.290 mL/min 0.066 mL/min 0
3xSystem 5xColumn
trr = = (3V (3VT T+ + 5V 5Vcc)/F )/F

where: where: t trr is is the the re-equilibration re-equilibration time time in in minutes, minutes, V VTT is is the the total total system system volume, volume, V Vcc is is the the column column volume volume in in mL mL F F is is the the flowrate flowrate in in mL/min. mL/min. column r column volume volume = = 0.7( 0.7( r22L/2 L/2) ) system system volume volume = = 650-3000 650-3000 L L
10
Volume, mL
10
20
30
40
Time, min
Reduction of Re-equilibration Time

Reduce Re-equilibration time, two Approaches:
Final Hold

(Approach (Approach 1 1- Increase Increase Flow Flow Rate) Rate)
Final Hold Final Hold
1. increase flow rate

Gradient Time Re-equilibration Total Cycle Time % Organic
nt
Gr
Gr
nt die G ra
ie ad
Inject
ie ad
Inject
Re-equilibration % Organic
nt
Inject
2. reduce column volume
Gradient Time
Re-equilibration
AU
% Organic % Organic
Total Cycle Time
1
Final Hold
2
Final Hold
Column: 3.9 X 50 mm Column volume (c.v.) =0.60 mL 5 minute gradient @ 1 mL/min instrument delay volume (d.v.) = 650 L gradient volume = g t x c.v. = 3
Column: 3.9 X 50 mm
Column volume (c.v.) =0.60 mL 5 minute gradient @ 2 mL/min instrument delay volume (d.v.) = 650 L g x c.v. = 3 gradient volume = t
Gr
Reduction of Re-equilibration Time (Approach 1 - Increase flow rate)

Symmetry C18
Alkylphenones
ad Gr
nt die G ra
ie ad
Inject
Gradient Time
Inject
Gradient TimeRe-equilibration Total Cycle Time
Total Cycle Time
Total re-equilibration time, rt Total re-equilibration time, rt = {3(0.65) + 5(0.7)(0.6)}/1 = {3(0.65) + 5(0.7)(0.6)}/2 = 4.0/1 = 4.0/2 = 4.0 min. = 2.0 min.
nt
re-equilibration time is reduced by 50%

(Approach (Approach 2 2 -- Reduce Reduce Column Column Volume) Volume)
Final Hold
Final Hold
ad Gr
3.9 x 50 mm 40-100%B 3.0 min, 3 mL/min 4.5 min, 2 mL/min 9.0 min, 1 mL/min
0 2 4 Minutes 6 8
Inject
t ien
t ien
Gradient Time
Inject
Gradient Time
Re-equilibration
% Organic
Total Cycle Time
Total Cycle Time
Column: 2.1 X 50 mm Column volume (c.v.) = 0.170 mL 5 minute gradient @ 1 mL/min instrument delay volume (d.v.) = 650 L gradient volume = t g x c.v. = 0.85 Total re-equilibration time, t r = {3(0.65) + 5(0.7)(0.17)}/1 = 2.5 min.
Column: 2.1 X 20 mm Column volume (c.v.) = 0.069 mL 5 minute gradient @ 1 mL/min instrument delay volume (d.v.) = 650 L gradient volume = t g x c.v. = 0.35 Total re-equilibration time, t r = {3(0.65) + 5(0.7)(0.069)}/1 = 2.0 min.
re-equilibration time is reduced by 20%
Reduction of Re-equilibration Time (Approach 2 - Reduce Column Volume)

1.20
Outline
Introduction Strategies for Higher Throughput Gradient Separations to Achieve Maximum Throughput and Optimal Resolution
system solutions reduce gradient delay volume decrease re-equilibration re-equilibration time reduce injection cycle time method solutions
2.1 x 50 mm
1.10
1.00
2.1 x 20 mm
0.90
Conditions: Symmetry C18, 5 m Mobile phase: A=0.1% TFA in water, B=0.1% TFA in acetonitrile Gradient: 0-60% B in 5 minutes Column temperature: 30.0 C Detector: 254 nm Injection volume: 1 L Flow rate: 1 mL/min.
0.80
0.70
0.60 AU 0.50
0.40
-Increase throughput by approximately 25% by reducing column volume from 0.170 (50 mm length) to 0.069 (20 mm length).
0.30
0.20
0.10
0.00 0.00 0.50 1.00 1.50 2.00 2.50 Minutes 3.00 3.50 4.00 4.50 5.00
Faster Chromatography Shorter Cycle Time

Reducing Total Cycle Time
Reduce Cycle Times by:
Programming a system purge in the method which occurs during the injection of the sample or... Employing two columns and performing column switching.
0
Samples per day 320 +16%
AU
276
Column: 3.9 X 50 mm 3 mL/min 3 min. gradient
Minutes
24
Higher Throughput Through Column Switching

Column: Symmetry, C18, 5 L, 19 X 50 mm Flow Rate: 20 mL/min. Re-equilibration requires 5 column volumes = 150 mL = 7.5 min. Re-equilibration period = unused time
Summary - System Solutions

Reducing Gradient Delay Volume Use 0.12 mm (0.005") i.d. tubing instead of 0.25 mm (0.009") to reduce system volume; Shorten all tubing lengths; Reduce the extra-column volume in the auto-injector by employing a smaller loop
Inject Gradient Hold Re-equilibration

Column switching can reduce runtimes by approx. 30%
0
1 column 2 columns
10
12.5
20
0 hrs
10.5hrs
16.5hrs
Note: a second pump must be employed
Remove gradient mixers
Summary - System Solutions (cont'd)

Achieve Faster Gradient Chromatography By...
Reducing Re-equilibration Time Reduce Reduce column volume volume Increase flow rate Reducing Cycle Time Program injection injection to occur occur during during re-equilibration re-equilibration Implement Implement column switching switching
Outline
Introduction Strategies for Higher Throughput Gradient Separations to achieve maximum throughput and maximize resolution system solutions method solutions use use shorter shorter gradients gradients use use higher higher flow flow rates rates use shorter columns use shorter columns use use smaller smaller particle particle sizes sizes
increase temperature
Optimizing Separations
The following diagram illustrates the cycle time parameters that were optimized to achieve high throughput goals.
Final Hold
% Organic
Factors Influencing Resolution in Gradient RP-HPLC Separations...

Resolution, Resolution, Rs, Rs, between between two two closely closely resolved resolved analytes analytes in in gradient RP-HPLC is a function of column efficiency gradient RP-HPLC is a function of column efficiency N, N, selectivity selectivity a a,, and and the the retention retention factor: factor:
Rs =
t w
N 4
ln
1 Bct0 + 1
Efficiency Efficiency Selectivity Selectivity Retention Retention

Gr ad ien t
c = %B/minute =
% tg
1 B.
Re-equilibration
% . t0 + 1 tg
Inject
Upon substitution of the actual variables ( %/tg (gradient time)) for c, gradient slope, one can see the relationship between gradient time and resolution, and....
What Factors Influence Gradient RP-HPLC Separations...

...further derivatization of this term shows the relationship between resolution and flow rate, F, and column length, L, or column volume, r22L/2.
Resolution Dependance on Gradient Time and Flow Rate for a Gradient Method
(Symmetry ,, 4.6 (Symmetry C C18 4.6 X X 50 50 mm, mm, 5 5 m) m) 18
Rs =
20
t w
N 4
ln
1 B. % tg
. . or2 .L/F + 1
t
Rs =
t w
N 4
ln
1 B. % . t0 + 1 tg
Rs
18 16 14 12 10 16 8 8 6 4 4 Gradient Time [min] 2 0 0.3 0.5 1 2 Flow [mL/min] 5 8 1
} }
Efficiency Selectivity Retention

1 It is the effect of these variables, % F, tgg and L, that we will investigate. B . tg . t
1. Effect of changing gradient run time, tg 2. Effect of changing flow rate, F
. r2 .L/F + 1
Impact of Reducing Gradient Time (tg g) on Resolution

Conditions:
Absorbance
Summary Impact of Gradient Time on Resolution
Gradient Time: 8 minutes

Column: Symmetry C18, 5 m, 4.6 X 50 mm Mobile phase: A=0.1% TFA in water, B=0.1% TFA in acetonitrile Gradient: 0-60% B in noted gradient time Column temperature: 30.0 C Flow rate: 1 mL/min. Detector: 254 nm Injection volume: 1 L
Minutes
5.50

Absorbance
Resolution increases as gradient time increases Throughput decreases as gradient time increases
Minutes
3.50

Absorbance
-Longest gradient time provides best resolution -Shortest gradient time maximizes throughput
Minutes 2.50
Gradient Time: 1 minute

Absorbance
-Reducing just gradient time sacrifices resolution
Minutes
2.50
Impact of Flow Rate (F) on Resolution

Flow rate: 1 mL/min.
Absorbance
Summary Impact of Flow Rate on Resolution
Conditions: Column: Symmetry C18, 5 m, 4.6 X 50 mm Mobile phase: A=0.1% TFA in water, B=0.1% TFA in acetonitrile Gradient: 0-60% B in 4 minutes Column temperature: 30.0 C Detector: 254 nm Injection volume: 1 L
Minutes
5.00
Resolution goes through an optimum due to the combination of gradient expansion and decrease in plate count Optimum resolution is approximately 1 to 2 mL/min for most practical separation problems

Absorbance
Minutes
3.50

Absorbance
- Resolution goes through an optimum due to the combination of gradient expansion and decrease in plate count
Minutes
2.50
Resolution Dependance on both Flow Rate and Gradient Time for a Gradient Method
(Symmetry , 4.6 (Symmetry C C18 18 4.6 X X 50 50 mm, mm, 5 m)
Absorbance
1.20
Reduction of Cycle Time

Flow rate: 0.5 mL/min. Grad. time: 16 min.
Conditions: Column: Symmetry C18, 5 m, 4.6 X 50 mm Mobile phase: A=0.1% TFA in water, B=0.1% TFA in acetonitrile Column temperature: 30.0 C Detector: 254 nm Injection volume: 1 L
Rs =
2 0 1 8
t w
1 N ln % . . 4 t or2 .L/F + 1 B. tg

16 14
Absorbance
} }
1.00
Flow rate: 1.0 mL/min. Gradient time: 8.0 min.
Minutes
14.00
12
10
Rs
16
8
0.60
Absorbance
4
2
Gradient Time [min] 2

0
3. Effect of changing gradient run time, tg, and flow rate, F
Flow rate: 2.0 mLmin. Gradient time: 4.0 min.
Minutes
9.00
Flow rate increased proportional to gradient time decrease. Elution pattern is maintained as cycle time is decreased resulting in an increase in throughput.
0.3 0.5 1 2 Flow [mL/min] 5 8 1
0.20
Absorbance
Flow rate: 5.0 mL/min. Gradient time: 2.0 min.
Minutes
3.50
Minutes
2.00
Summary Reduction of Cycle Time

Introduction Introduction
Outline
Strategies Strategies for for Higher Higher Throughput Throughput Gradient Gradient Separations Separations to achieve maximum throughput and maximize achieve throughput and maximize resolution resolution system system solutions solutions
To obtain the maximum sample throughput the gradient time must be adjusted inversely proportionally to the flow rate. As shown in the previous slide the sample throughput was increased by 800% upon increasing the flow rate to 5 mL/min. and reducing the gradient time to 2 minutes.
method method solutions use use shorter gradients gradients use higher flow use flow rates use use shorter columns columns use smaller particle use particle sizes sizes increase increase temperature temperature
10
The Number of Column Volumes per Minute Impacts Resolution
Impact of Column Length on Resolution

2 Approaches:
How Short is Too Short?

It is not the column length which influences the separation in so much as the number of gradient volumes moving across the column.
Approach 1: Gradient volume in not proportion to the column volume (gradient run time constant while changing the column length). Approach 2: Scale gradient volume in proportion to the column volume (change the gradient run time proporionally with the column length).
11
Column Volume to Gradient Volume Relationship (Approach 1) -Gradient volume not scaled scaled to column volume
50 mm column 20 mm column
Impact of Column Length on Resolution (Approach 1)
-Gradient Gradient volume volume not scaled scaled to to column column volume
,2.1 x 20 mm
2.1 x 30 mm
1.20 1.10
Conditions: Symmetry C18, 5 m Mobile phase: A=0.1% TFA in water, B=0.1% TFA in acetonitrile
2.1 x 50 mm
Gradient: 0-60% B in 5 minutes

Column temperature: 30.0 C Detector: 254 nm Injection volume: 1 L Flow rate: 1 mL/min.
Column volume = 0.5 mL 5 minute gradient @ 1 mL/min gradient volume = tg x f.r. = 5 Total volume = g.v./c.v. = 10 column vols.
Column volume = 0.2 mL 5 minute gradient @ 1 mL/min gradient volume = tg x f.r. = 5 Total volume = g.v./c.v. = 2 column vols.
AU
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
Maintain resolution by not scaling gradient volume proportionally to column volume. However maximun reduction of analysis time is not realized as when gradient volume is scaled.
0.10
0.00 0.00 0.50 1.00 1.50 2.00 2.50 Minutes 3.00 3.50 4.00 4.50 5.00
Column Volume to Gradient Volume Relationship (Approach 2) -Gradient Gradient volume volume scaled scaled to to column column volume volume
50 mm column 20 mm column
What Factors Influence Gradient RP-HPLC Separations...

L (column length) is varied. Gradient volume is scaled in proportion to the column volume.
Column volume = 0.5 mL
Column volume = 0.2 mL
5 minute gradient @ 1 mL/min

gradient volume = tg x f.r. = 5 Total volume = g.v./c.v. = 10 column vols.
2 minute gradient @ 1 mL/min

gradient volume = tg x f.r. = 2 Total volume = g.v./c.v. = 10 column vols.
Rs =
t w
N ln 4
1 B. % . t tg
. r2 .L/F + 1
Efficiency Selectivity
Retention
Terms are constant
}
12
Impact of Column Length on Resolution (Approach 2) -Gradient volume scaled to to column volume
2.1 x 10 mm Gradient time = 1 minute 2.1 x 20 mm Gradient time = 2 minutes 2.1 x 30 mm Gradient time = 3 minutes
1.20 1.10 1.00 0.90 0.80
Summary Impact of Column Length on Resolution
2.1 x 50 mm Gradient time = 5 minutes
Conditions: Symmetry C18, 3.5 m Mobile phase: A=0.1% TFA in water, B=0.1% TFA in acetonitrile Gradient: 0-60% B in noted time Column temperature: 30.0 C Detector: 254 nm Injection volume: 1 L Flow rate: 1 mL/min.
0.70 0.60 AU 0.50 0.40 0.30 0.20
-Reduce analysis time by >50%. -Trade-off: reduction in resolution
Maximum sample throughput is realized when the gradient volume is scaled proportionally to the column volume.
0.10 0.00 0.00 0.50 1.00 1.50 2.00 Minutes 2.50 3.00 3.50 4.00 4.50
Gradient Delay Time

Final Hold
Gr ad ien t
t en di ra
Final Hold
Impact of the Number of Column Volumes on Peak Shape

0.05
% Organic
% Organic
Inject
Re-equilibration
Inject
Re-equilibration
2.1 x 10 mm Peak Width for Peak # 2 = 0.0533
Column: 4.6 X 50 mm Column volume (c.v.) = 0.83 mL 5 minute gradient @ 1 mL/min instrument delay volume (d.v.) = 650 L gradient volume = tg x c.v. = 6 Total system volume = 0.7(c.v.) + d.v. = 1.2 mL
Column: 2.1 X 50 mm
0.04
Column volume (c.v.) = 0.17 mL 5 minute gradient @ 0.2 mL/min instrument delay volume (d.v.) = 650 L gradient volume = tg x c.v. = 6 Total system volume = 0.7(c.v.) + d.v. = 0.72 mL
0.03
Conditions: Symmetry C18, 5 m Mobile phase: A=0.1% TFA in water, B=0.1% TFA in acetonitrile Gradient: 0-60% B in 4 minutes Column temperature: 30.0 C Detector: 254 nm Injection volume: 1 L Flow rate: 2 mL/min.
AU 0.02
Column re-equilibration time = total vol./f.r. Column re-equilibration time = total vol./f.r. = 0.67/0.2 = 1.15/1 = 3.6 min. = 1.2 min.
0.01
gradient is delayed by a factor of 3

0.00 0.20 0.40 0.60 0.80 1.00 Minutes 1.20 1.40 1.60 1.80 2.00
13
Reducing the Effect of Gradient Delay Delay Volume

- Make Make gradient gradient steeper steeper by by increasing increasing the the flow flow rate rate or or decreasing decreasing the the gradient gradient
time time
Outline
Introduction Introduction Strategies Strategies for for Higher Higher Throughput Gradient Separations to achieve achieve maximum maximum throughput throughput and maximize resolution resolution system system solutions
0.05 0.04 0.03 AU 0.02 0.01 0.00 0.20
peak width for peak # 2 = 0.0533
0.40
0.60
0.80
1.00 Minutes
1.20
1.40
1.60
1.80
2.00
Increase flow rate (2 to 3 mL/min.)
Increase gradient slope by 50% (4 to 2 min. grad)
0.030 0.025 0.020 AU 0.015 0.010 0.005 0.000
method solutions use shorter gradients gradients use higher flow rates use shorter columns columns use smaller particle sizes increase temperature
2.00
0.20
0.40
0.60
0.80
1.00 Minutes
1.20
1.40
1.60
1.80
2.00
0.20
0.40
0.60
0.80
1.00 Minutes
1.20
1.40
1.60
1.80
Impact of Particle Size (dp) on Resolution
Comparison of Resolution Dependence on Particle Size

Symmetry C18, 3.5 m, 4.6 X 50 mm Max. Resolution @ 1 mL/min., 16 min. 20 gradient = 18.8 18
16 14 12 Rs Rs 10 8 6 4 2 0 0.3 2 0.5 4 Gradient Time [min] 8 6 4 2 0 0.3 2 0.5 1 Flow [mL/min] 4 Gradient Time [min]
AU
1.20 1.10 1.00 0.90 0.80 0.70 AU 0.60 0.50 0.40 0.30 0.20 0.10 0.00 0.00
1.00 0.90
Conditions:
Symmetry C18, 5 m, 4.6 X 50 mm Max. Resolution @ 1 mL/min., 16 min. 20 gradient = 16.2

18 16 14 12 16 10 8 8 16
Symmetry C18, 3.5 m, 2.1 x 50 mm Rs (peak A and B) = 11.97 Rs (peak B and C) = 8.79 Rs (peak C and D) = 7.35 C A
0.50 1.00 1.50 2.00
B
Minutes 2.50 3.00 3.50 4.00 4.50
Columns: Symmetry 1 C 8 , 5 m, 4.6 8, 3.5 m, X 50 mm and Symmetry 1C 4.6 X 50 mm Mobile phase: A=0.1% TFA in water, B=0.1% TFA in acetonitrile Gradient: 0-60% B in 4 minutes Column temperature: 30.0 C Detector: 254 nm Injection volume: 1 L Flow rate: 1 mL/min.
0.80 0.70 0.60 0.50 0.40 0.30 0.20
18, 5 m, 2.1 x 50 mm Symmetry C Rs (peak A and B) = 6.71 Rs (peak B and C) = 7.32 Rs (peak C and D) = 6.45 C
-Achieve increased resolution with the smaller particle size material in the same gradient time -Increase throughput and resolution with smaller particle size if flow rate is increased
B A
0.50 1.00 1.50 2.00 2.50 Minutes 3.00 3.50 4.00 4.50 5.00
1 Flow [mL/min]
1 5
1 8
0.10 0.00 0.00
14
Summary Impact of Particle Size on Resolution
Outline
Introduction Introduction Strategies Strategies for for Higher Higher Throughput Throughput Gradient Gradient Separations Separations to to achieve achieve maximum maximum throughput throughput and and maximize maximize resolution resolution system system solutions solutions method method solutions solutions use shorter gradients use higher flow rates use shorter columns use smaller particle sizes increase temperature
Resolution is increased as a result of using a smaller particle size. This is due to the increase in the number of theoretical plates. If the flow rate is increased as well as the particle size being decreased, an increase in sample throughput is realized with increasing resolution.
Impact of Temperature
Faster Separtation Narrower Peaks Lower Backpressure Lower Viscosity Greater Sensitivity-
Summary - Method Solutions

To obtain the fastest throughput:
increase flow rate decrease column volume decrease particle size scale gradient volume with decrease in column volume increase temperature to reduce viscosity of mobile phase allowing increases in flow rate
50 40 30 AU 25 C
2 3 Minutes
15

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Gradient Operation in HPLC

Dr. Shulamit Levin Medtechnica

Gradient Operation in HPLC

Introduction - Optimizing Gradient Separations

Dr. Shulamit Levin Medtechnica

Gradient Time Total Cycle Time

Typical Problems Encountered in Gradient Chromatography

Dr. Shulamit Levin, Medtechnica

Introduction - Options to Improve Sample Throughput

Test Probe Structures

Initial Separation and Conditions

0.18 0.16 0.14

0.12 0.10 AU 0.08

Detector: 254 nm Injection volume: 1 L

A - 1-hydroxy-7-aza-benzotriazole B - 4-methylbenzene sulfonamide C - methyl 3-amino-2-thiophenecarboxylate D - 4-aminobenzophenone

H. Weller, Bristol-Myers Squibb Pharmaceutical Research Institute

Dr. Shulamit Levin, Medtechnica

Volumes in an HPLC System

Determination of System Precolumn Volume

Column Volume Column Detector

1.0 0.8 0.6

Extra Column Volume

System components affecting dwell volume: Pump Gradient Mixers Injector

Effect of Precolumn Volume

Gradient Shape and Precolumn Volume

Total Cycle Time

Total Cycle Time

Dr. Shulamit Levin, Medtechnica

Gradient Shape and Re-equilibration

trr = = (3V (3VT T+ + 5V 5Vcc)/F )/F

Dr. Shulamit Levin, Medtechnica

Reduction of Re-equilibration Time

Reduction of Re-equilibration Time

1. increase flow rate

Gradient Time Total Cycle Time

2. reduce column volume

Dr. Shulamit Levin, Medtechnica

Total Cycle Time

Reduction of Re-equilibration Time (Approach 1 - Increase flow rate)

Gradient TimeRe-equilibration Total Cycle Time

Total Cycle Time

re-equilibration time is reduced by 50%

Reduction of Re-equilibration Time

Total Cycle Time

Total Cycle Time

re-equilibration time is reduced by 20%

Reduction of Re-equilibration Time (Approach 2 - Reduce Column Volume)

Faster Chromatography Shorter Cycle Time

Samples per day 320 +16%

Dr. Shulamit Levin, Medtechnica

Higher Throughput Through Column Switching

Summary - System Solutions

Inject Gradient Hold Re-equilibration

Note: a second pump must be employed

Remove gradient mixers

Summary - System Solutions (cont'd)

Dr. Shulamit Levin, Medtechnica

Factors Influencing Resolution in Gradient RP-HPLC Separations...

Efficiency Efficiency Selectivity Selectivity Retention Retention

Gradient Time Total Cycle Time

What Factors Influence Gradient RP-HPLC Separations...

18 16 14 12 10 16 8 8 6 4 4 Gradient Time [min] 2 0 0.3 0.5 1 2 Flow [mL/min] 5 8 1

Efficiency Efficiency Selectivity Selectivity Retention Retention