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This is one of the most common questions that I hear, especially when training people with less experience in an analytical laboratory setting. Unfortunately, there is not a single source of information or recommendations that can provide an answer for every user. Usually, each method provides instructions on how and when to calibrate, so let’s look at some of the reasons for these decisions.
Why do we calibrate?
First, we need to discuss the reason for needing to calibrate. Methods and instruments do not generate quantitative answers automatically.
Every analytical instrument requires some form of calibration.
Using materials of known purity, we prepare one or more standards, each containing a known amount of the substance that we are trying to measure. We can now relate the response from the system with the amount of substance in the standard. Mathematically, this means:
Response = RF * Amount.
Once we know the Response Factor (RF), we can calculate the Amount in an unknown if we know the response produced by that sample. The response factor is sometimes referred to as a calibration factor, or you could think of it as a conversion factor from instrument response units to sample amount units.
Every instrument needs a response factor.
How do we calibrate?
There are several ways to develop the calibration factors for a particular method with a particular compound. Options include external standards, internal standards, and process standards. For each of these options, we can calibrate using only a single level, or we can prepare standards containing different amounts, and construct a plot of response against the amount of sample (i.e., a calibration curve). The reasons for choosing a particular strategy are beyond the scope of this discussion. We will discuss them in a separate post.
How often should I calibrate my HPLC?
The general philosophy of Quality Control (QC) for chromatographic analysis states that a sample analysis is valid only if the system has been calibrated or the calibration has been checked recently. If the system fails calibration (or a calibration check), then any future sample analyses will not be valid. Furthermore, all results obtained since the last successful calibration are now possibly incorrect, since we do not know exactly when the instrument failed.
How often you calibrate will depend on how often you want to allow such a failure. Put another way,
your calibration frequency is all about RISK! How much risk are you willing to accept that the instrument drifts out of calibration and you do not recognize the problem? Let’s look at how different laboratories manage this risk.
High-Value Analysis Requires Low Risk
I often use the pharmaceutical industry as an example of this situation. The analytical lab prepares calibration standards for each analysis batch, makes multiple injections of the standards, and multiple injections of the sample. The analysis sequence will have calibration standards, blanks, and system suitability (to verify performance) followed immediately by samples. Additional standards are injected after the samples. These “bracketing” standards are sometimes included in the calibration calculation or they may be used as calibration verification. This approach satisfies the general QC philosophy that we mentioned above but minimizes the risk of a calibration-related error.
Wide Specifications Allow More Risk
In some industries, the acceptable ranges for a compound may be relatively large (+/- 10 % or more). Since product quality and performance are not significantly affected by these changes, the analytical requirements can be adjusted to allow less frequent calibration. Typical calibration schedules can range from once a day to once a month, depending on the relative risk levels. I see these situations in petroleum laboratories, chemical additives manufacturers, and some parts of the food industry. I have even visited laboratories that only recalibrate their gas chromatographs once a year! However, I do not recommend this schedule for HPLC.
So, what works for your laboratory? How would you make that decision?
In the next FB post, we will discuss some of the specific considerations that go into deciding which schedule is best for you. If you have questions about any of these general topics, please post them here.
If you have any other questions or concerns, feel free to reach out to us or post on the Facebook link below.
