Waters Alliance LC/MS System
Effect of Temperature on Column Pressure,
Column backpressure
Peak shape
Peak retention time
Key Words
Temperature, pressure,
viscosity, peak shape,
sensitivity
AMD30 1999 Waters Corporation
Peak Retention Time and Peak Shape
Jeanne B. Li, Waters Corporation, Milford, MA
Background
To optimize chromatography for high throughput applications, the use of
columns with 2-3 micron particles and higher than normal flow rates results in
high pressures. Increasing temperature can reduce the backpressure. This
application note illustrates some of the effects of temperature on column
pressure, peak retention time and shape.
Column backpressure as a function of solvent composition
The two most common mobile phases for reverse phase chromatography are
methanol-water and acetonitrile-water. The viscosity of the mobile phase
changes with the composition. The increase in viscosity increases pressure.
The maximum pressure can be significant for 50% methanol-water.
Pressusre vs. Organic Solvent
1600
1400
MeOH
1200
ACN
Figure 1. Pressure vs. Organic
1000
Solvent Composition
Column: Symmetry C18, 2.1x 30mm
psi
800
600
Flow rate: 0.3 mL/min
Temperature: Ambient (ca. 20 C)
400
200
0
0 20 40 60 80 100
Percent Organic Solvent
The LC/MS Market Development Group 1
 Column backpressure as a function of flow rate and temperature
Pressure increases linearly with flow rate. The solvent viscosity determines the amount of pressure. This is shown in
Figure 2. As the temperature increases, the viscosity decreases and the column backpressure will decrease. This is
shown in Figure 3. The pressure can be reduced as much as 43% by raising the the temperature from 20 to 60C. This
allows higher flow rates to be used. In addition, the lower pressures can reduce wear on the HPLC instrumentation and
can possibly increase column life.

Application to high throughput

Pressure is also linearly proportional to column length for the same column diameter and packing (data not shown).
Therefore, for the fastest injection-to-injection cycle times, a method should use a short column, an elevated temperature
and an increased flow rate. See LC/MS Applications Notes AMD21 and AMD22.

P re s s u re v s . F lo w Ra te a t 2 0 C P re s s u re v s . T e m p e ra tu re
5000 5000

50 % M e O H 50% MeO H
4500 4500
50 % A C N 5 0 % AC N
100% H2O
10 0% H 2O
4000 4000 100% MeO H
10 0% M eO H
1 0 0 % AC N
10 0% A C N
3500 3500

3000 3000
psi

psi
2500 2500

2000 2000

1500 1500

1000 1000

500 500

0 0
0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 1 0 .0 2 0 .0 3 0 .0 4 0 .0 5 0 .0 6 0 .0 7 0 .0
m L /m in D e g re e s , C

Figure 2. Pressure vs. Flow Rate Figure 3. Pressure vs. Temperature

Column: Symmetry C18, 2.1x 30mm Column: Symmetry C18, 2.1x 30mm
Flow rate: 0.2 to 1.0 mL/min Flow rate: 1.0 mL/min
Temperature: 20 C Temperature: 20C to 60C

AMD30 1999 Waters Corporation The LC/MS Market Development Group 2

 Effect of temperature on peak retention time and peak shape
As the temperature is increased, the peak retention times are shortened. Chromatography is a series of equilibrium reactions
where the analytes are either dissolved in the mobile phase or adsorbed to the stationary phase of the column. The higher the
temperature, the faster the exchange of the analytes between the mobile phase and the stationary phase. This is seen in
Figure 4. A simple isocratic separation show that a 30% reduction in retention time is the result of elevating the temperature to
50C.

The overlay of Peak 4 of the 25 C and 50 C chromatograms shows the peak height is greater and the peak is narrower. The
results is an increase in sensitivity of approximately 15%.

Peak 4
50C
50
40
25C
30

AU 25 C

0 1 2 3 4
Minutes
Figure 4. Effect of temperature on retention time and peak shape
Isocratic separation of acetone a C2-C4 alkylphenones in 60:40 methanol-water on a Symmetry C18 2.1x30mm column

AMD30 1999 Waters Corporation The LC/MS Market Development Group 3