Part 4: Operating Conditions
If you are writing the perfect method (or at least trying), this the most important part, right? Just write down the instrument settings (assuming this is a chromatographic method), and you are done. But make sure that all critical information is included.
Each module/component has settings, and your method must include the important parameters for each. LC systems have pumps, autosamplers, column ovens, and detectors. GC systems have the same components, although the settings can be more complex when it comes to injection. The following checklist can be used to verify that you have everything. This list is easily prepared by having the instrument control software displayed when you write the method - if it is in the software, it needs to be in the method.
Do:
- You must include the following information for LC methods:
- Flow rate , mL/min.
- Injection volume, in uL, and other settings such as wash solvents, times, locations, etc., and any other special operating modes (low delay volume mode, overlapped injection cycles, etc.)
- Column oven temperature, in degrees C
- Detector wavelength, bandwidth (for diode array), reference settings (for diode array if needed), slit width, data acquisition rate (in Hz or by peak width), and time constant/response time. These last two are often not included, but can profoundly affect your results if not set correctly.
- Equilibration conditions and time
- You must include the following information for GC methods:
- Carrier gas (we covered purity in a previous section)
- Inlet temperature (liner size and style comes under Equipment)
- Injection volume, pre- and post-wash settings and solvents, number of pumps, delay/equilibration times (as needed)
- Injection technique (split, splitless, cold on-column, etc) and all settings for that technique (times, flows, pressures, etc.). If it is in your method setup screens, it must be in the method.
- Oven temperature and complete details for temperature programming
- Equilibration time and conditions
Do Not:
- Specify instrument-specific settings, unless the method is validated for and expected to be only used on that brand of equipment.
- Offer alternative settings or ranges for settings unless you have evaluated these changes.
Next Month: Preparation of Standards
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Doing "Too Much" in the Lab
When I visit most labs, it is common to note that there are several things that they are not doing, or not doing often enough. However, sometimes I see labs that are trying too hard to do the right thing, or to prevent problems. Sometimes, doing too much means that the lab is just wasting money and/or time, but in other situations, you can actually cause more problems. Some examples are given below:
- Filtering HPLC-grade Mobile Phases
- Filtering of high purity solvents is only needed if you have dissolved a solid in the solvent. Otherwise, the process and transfers needed to complete the filtering are more likely to introduce new contaminants.
- Degassing Solvents by Vacuum Filtration, But Not Maintaining the Degassed State
- This was a common practice 20 years ago (before on-line degassers), but after filtering you always maintained a constant positive pressure of Helium in the container. If you just pour the filtrate into the mobile phase bottle, it begins to re-gas immediately.
- Using Long Equilibration Times
- Some labs will equilibrate their systems at starting conditions for one hour before use. There is usually no reason for this time. Fifteen minutes is usually sufficient for a modern instrument and column. A better option would be to include at least one programming blank (no injection) or one blank injection, and at least one standard injection before beginning the analysis. Both of these options will better prepare the system for analysis.
Don't be afraid to challenge "the way we have always done it."
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Quote for the Day
"Everybody is ignorant only on different subjects."
– Will Rogers,in P. M. Love, The Will Rogers Book,
p. 199 (1972)
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Dowload Recent Presentations
Get your own copy of two recent presentations. Both cover our recent work on HILIC columns. These publications describe a simple way to evaluate multi-mode mechanisms on HILIC columns.
Pittcon, March, 2014:
Mixed Mode Mechanisms in LC: Curse or Cure?
Minnesota Chromatography Forum, May, 2014:
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Vendor Seminars
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The following courses are offered by Agilent Technologies at their Atlanta, GA training center.
Infinity HPLC Maintenance and Troubleshooting
June 17-18, 2014 (seats available)
August 20 - 21, 2014 (seats available)
October 28 - 29, 2014 (seats available)
December 16 - 17, 2014 (seats available)
Learn how to maintain your 1220, 1260, and 1290 instruments.
HPLC OpenLab ChemStation Operation
July 15 - 18, 2014 (seats available)
September 23 - 26, 2014 (seats available)
December 2 - 5, 2014 (seats available)
Learn all the features of this new software program in a hands-on setting.
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Contact Us
Merlin K. L. Bicking, Ph. D.
ACCTA, Inc.
3534 Jessie Ct
Saint Paul, MN USA 55125
Email: info@accta.com
Internet: www.accta.com
Phone:651-731-3670
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