Waters ACQUITY APC Columns - Manual (Page 5)

5
ACQUITY APC Columns
Table 4. Viscosity of Common Solvents at
Different Temperatures **
Solvent
Viscosity (cP)
20 °C 40 °C
Acetone 0.32 0.26
Chloroform 0.57 0.45
Dimethyl formamide (DMF) 0.92 0.68
Dimethyl sulfoxide (DMSO) 2.00 1.50
Ethyl Acetate 0.44 0.36
Hexane 0.33 0.26
Methanol 0.60 0.44
Methylene chloride 0.44 N/A
N-Methylpyrrolidone (NMP) 1.70 1.30
Tetrahydrofuran (THF) 0.55 0.39
Toluene 0.59 0.46
Water 1.00 0.67
d. Minimizing Band Spread
The ACQUITY APC System was designed to minimize band
spreading. Deviation from Waters specified tubing could result
in deterioration of chromatographic performance. Figure 5
shows the influence of tubing inner diameter. Using larger
tubing causes excessive peak broadening, lower sensitivity,
and loss in resolution.
Diluted/Distor ted Sample Band
0.005 inches
0.020 inches
0.040 inches
Figure 5. Effect of Connecting Tubing on System.
IV. TROUBLESHOOTING
Changes in retention time, resolution or backpressure are
often due to column contamination. See the Column Cleaning,
Regeneration and Storage section of the care and use manual.
Information on column troubleshooting problems may be
found in HPLC Column Theory, Technology and Practice, U.D.
Neue, (Wiley-VCH, 1997); Waters HPLC Troubleshooting Guide
(Literature Code # 720000181EN); or by visiting www.waters.com
V. COLUMN CLEANING, REGENERATION
AND STORAGE
Assuming that there is no damage to the column bed, changes
in peak shape, peak splitting, shoulders on the peak, shifts in
retention, change in resolution or increasing backpressure
may indicate contamination of the column. Flushing with high
concentrations of organic solvent, taking care not to precipitate
buffers, is usually sufficient to remove the contaminant. If the
flushing procedure does not solve the problem, purge the column
using the following cleaning and regeneration procedures.
a. Cleaning and Regeneration
Use a cleaning routine that matches the properties of the samples
and/or what you believe is contaminating the column. The
columns are stable using a wide range of solvents to improve the
dissolution of the contaminant. Before beginning any cleaning
procedure it is best to isolate the column from downstream
For example, if you were to select a three-column bank using
45Е, 200Е and 450Å columns and require that DMF to be used
as the mobile-phase solvent, you would be limited to a maximum
flow rate of 1.0 mL/min. In this case, DMF has a solvent viscosity
of 0.92 cP at 25 °C. Under these conditions the 450Å column
limits the flow to 1.0 mL/min, even though the other columns
can support a higher flow.
Note: When connecting multiple columns in series, the maximum flow rate may
not be achievable due to pressure limitations of the instrumentation. Please
refer to the system's owner manual for more information.
Table 3. Recommended Mobile Phase
Flow Rate for a Single ACQUITY APC Column
Pore Size
(Å)
Maximum Flow
Rate at Solvent
Viscosity < 0.6 cP
Maximum Flow
Rate at Solvent
Viscosity > 0.6 cP
45 1.8 mL/min 1.1 mL/min
125 2.0 mL/min 1.6 mL/min
200 2.0 mL/min 1.4 mL/min
450 1.7 mL/min 1.0 mL/min
900 0.90 mL/min 0.60 mL/min
**References:
1. Reid, R.C., Prausnitz, J.M., Poling, B.E. The Properties of Gases and Liquids,
4th Edition, McGraw Hill, 1987, Table 9-8.
2. http://www.wolframalpha.com
3. Yang, J. Chem Eng Data (2008), 53, 1639-1642.