You know those pieces of paper that you sometimes run across when you open a box containing a new column? Maybe you don’t have much time to look at it or it sometimes gets lost along the way. Let’s stop for a minute and look at what that information is and how it pertains to your projects in the lab. For starters, our LC column testing is actually a multi-step process. We start by testing essential chemistry requirements from a representative sample for each batch or lot number of packing material. Those results ensure the quality of that material before we use it to pack more columns. The second stage of testing is specific for each column that we pack, and results in the certificate that you see in the box with the finished product. Those results tell us how well that individual column was packed. Of course, if it does not meet our strict testing criteria, you will never see that column or certificate! Here are the important things to look for on the certificate:
Catalog number, Lot number, Serial number and “Tested” date
(Lot numbers identify packing material processed on a certain date, similar to a “batch”, whereas serial numbers are unique for each column.)
Retention time and specific parameter(s) from each of the reversed phase test mix marker compounds:
Uracil – Void Volume
Toluene –Hydrophobic Retention
Naphthalene –Hydrophobic Retention
Biphenyl –Overall Hydophobicity, Asymmetry and EfficiencyFlow rate and Pressure (with mobile phase listed)
Every HPLC or UHPLC column is tested under optimal conditions with a column test mix standard, usually with the LC Reversed Phase (RP) Test Mix (Please see the example certificate below). Each compound is included for a specific reason, which I will describe briefly.
Uracil is a very polar and rarely retained at all by reversed phase columns. For that reason, uracil is used to measure void volume. Be aware also that the internal volume of the instrument between the injection valve and detector also contributes to this volume, so you will likely see slight differences between different instruments. Columns are tested at Restek using configurations that minimize dead volume in tubing and connections, so chances are that your void volume will be slightly larger.
Toluene and naphthalene are used to monitor hydrophobic retention. You can get a good idea of column retention, as well as get a rough idea of resolution between the two at a quick glance.
Since it is the latest eluting peak, the retention time of biphenyl is used to measure overall hydrophobicity, column efficiency, and asymmetry. Hydrophobicity is the overall retention of the column, while efficiency is the number of theoretical plates. Asymmetry is a measurement of how much the shape varies from a perfect Gaussian curve, which is neither tailing nor fronting.
The best way to use a test mixture like this in your lab is to inject the mixture on your new column after equilibration. Doing so will allow you to gauge the combined performance of the LC system and the column. It is important to note that we take great strides to eliminate external factors and measure only the column performance for the QA Report. Because of the potential influence of the LC system, it can be expected that results will not match precisely. The test mixture can be analyzed again periodically, or when troubleshooting performance. Comparing the first results to any subsequent results may be useful in indicating issues with the LC system or column. Keep in mind that some changes are expected over the lifetime of a column when samples are being injected, but you may be able to recognize changes more readily by doing this.
Another helpful piece of information that should be collected from the above analysis is the pressure. You will most likely see some difference in pressure versus what it was when tested at Restek due to mobile phase differences and system pressure. However, if you notice a significant change after equilibration, you can go back to the original conditions and compare the pressure to what it was when you first installed it onto your system. Remember that the flow rate and solvent composition has an effect on pressure.
Getting back to the original question “Why do you need to keep the certificate?”, there are several reasons why this might be helpful. First of all, the certificate records when the column was tested by Restek, it also contains the catalog number, the lot number of packing material used, and the serial number. Should you have any questions or experience any issues, all of this information will be needed to supply you with the best possible service. You may also need this information for your records, as it may be required by regulatory bodies or you may need it to accurately distinguish one column from another.
A big thanks goes to my colleague in Innovations, Ty Kahler, for his contributions to this post.
Thank you for your interest.