Advertisement
[ CARE AND USE MANUAL ]
For example, if you were to select a three-column bank using
45Å, 200Å 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
Maximum Flow Rate
Pore Size
at Solvent Viscosity
(Å)
< 0.6 cP
45
1.8 mL/min
125
2.0 mL/min
200
2.0 mL/min
450
1.7 mL/min
900
0.90 mL/min
Table 4. Viscosity of Common Solvents
at Different Temperatures **
Solvent
Acetone
Chloroform
Dimethyl formamide (DMF)
Dimethyl sulfoxide (DMSO)
Ethyl Acetate
Hexane
Methanol
Methylene chloride
N-Methylpyrrolidone (NMP)
Tetrahydrofuran (THF)
Toluene
Water
**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.
Maximum Flow Rate
at Solvent Viscosity
> 0.6 cP
1.1 mL/min
1.6 mL/min
1.4 mL/min
1.0 mL/min
0.60 mL/min
Viscosity (cP)
20 °C
40 °C
0.32
0.26
0.57
0.45
0.92
0.68
2.00
1.50
0.44
0.36
0.33
0.26
0.60
0.44
0.44
N/A
1.70
1.30
0.55
0.39
0.59
0.46
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.
0.005 inches
0.020 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
0.040 inches
Diluted/Distor ted Sample Band
ACQUITY APC Columns
5
Advertisement
