Lab Queries: What to Do When Things Go Wrong

I’m sure you’ll all be happy to know that this week on the blog we are leaving calibration curves and math behind us!

Don’t worry, if you still have questions about instrument calibrations, or a new issue arises in your lab, I’m always here to help you address those.  In talking to some of you at FELC a few weeks ago, the issue of troubleshooting came up a few times.  I know this can be a hard topic and really digging into a broken instrument can be an intimidating idea for some people.  But I’m going to let you in on a secret….it’s maybe one of my favorite activities to do!

I’m weird….I know…..I’ve accepted it and moved on. 😊

What to Do When Things Go Wrong

We’ve all had that moment….we started samples on an instrument, left for the night expecting to come back in the morning to a nice, completed data set, and return to find that our instrument malfunctioned!  Now results are behind schedule and you have the added issue of an instrument that just refuses to cooperate.

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Troubleshooting can be a frustrating and tricky activity to complete, especially because instruments never break at convenient times.  If I had a dollar for every time an instrument broke when I had rush samples to do, I wouldn’t have to work anymore!  But, because instruments will always continue to break, and I need to keep my job, I thought I’d fill you in on some of my favorite troubleshooting tips.

  • Follow the Path

A good way to make sure you don’t overlook any parts of your instrument is to start at one end of the instrument and follow the sample path through to the other end.  It’s easy to jump from place to place first, maybe checking your sample loop before jumping up to check your eluent filters.  Odds are that you’ll over look something, and your issue will be in the place you over look.  Following the path helps ensure that you give every section of you instrument it’s due investigation.

  • The Tubing Checker

One of my new favorite troubleshooting tools is a spare autosampler syringe!  When I need to troubleshoot one of my instruments with a large amount of tubing, I’ve found that using the syringe to carefully inject DI water into each section of tubing is a quick and easy way to look for the source of the problem.  Maybe a section of tubing is plugged, maybe it’s cracked, maybe there’s a dust bunny lodged in the end (true life this has happened to my IC!!)  It’s so hard to tell sometimes by just eyeballing the sections, but this technique has yet to fail me!

  • Look Beyond the Science

Sometimes the source of an instrument issue isn’t related to the “science-y” components we would typically focus on.  Sometimes exhaust fans wear out or autosampler screws fall out or sheer off.  Don’t be afraid to look into the more mechanical parts of your instruments.  Think of them as support systems.  If your autosampler syringe housing is loose and wobbly because a screw is loose, maybe the syringe isn’t being held firm enough to puncture the vials.  If you’re lucky, your instrument will be smarter than you.  Sometimes the software might notice that something is wrong and stop the analysis run.  If it’s not….you’ll come back to a broken syringe plus a lack of data.

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If you have any fun or interesting troubleshooting tricks that have never steered you wrong let me know!  I’m always on the hunt for new ideas, and if I get enough, we might do a part 2 later.  Having as many tools in your troubleshooting tool belt as possible is so beneficial, and it’s one of the areas that we should all constantly be striving to improve.

Part 3: How to Know if Your Calibration Curve is Correct

You’ve bought some standards and you made a multi-point curve.  You might think your work to improve laboratory accuracy is over, but then doubt starts to creep into your mind.  What if something’s gone wrong!?!  What if you run a sample and you expect one answer and your instrument tells you another?!  Is the sample bad, or is there a problem with your calibration curve?  Has something gone wrong somewhere in your plant’s process, or do you have a bad batch of calibration standards!?

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The first step in ensuring that your calibration curve is up to par is checking the Correlation Coefficient.  I know, I know….more math….but stick with me here because it’s important!  The R2 value of your calibration curve has a large amount of statistical power.  No one wants to talk about statistics, but it has a lot to do with how well your instrument is calibrated.

In a nutshell, the correlation coefficient gives you an idea of how well the standards relate to each other.  R2 values statistically can range from -1.0 to +1.0, but on your instruments, you should look for a value as close to 1.0 as possible!  Values of R2=0.9987, R2=0.99943, or R2=0.9963 are examples of what you are looking for.  The closer to 1.0 your R2 value is, the stronger the relationship between your standards.  A strong relationship between standards leads to more confidence in the reported results for your samples.  Remember that your standard value points hug your samples, and no one likes crappy, wet noodle armed hugs! It’s listed somewhere in your instrument’s software, you might just have to look around for it.

So you’ve got a strong curve, but calibration curves can be strong….but wrong!  How can you check the accuracy of the standards you used to make the curve?

There is a way to prove that you standards and instrument are operating correctly.  We are going to employ the Dr. Sheldon Cooper of standard….the validation standard!  Just like Dr. Copper, the validation standard knows more than you, and it isn’t afraid to tell you when you’re calibration is wrong!

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The validation standard should be a standard SEPARATE from your curve!  Buy it from an alternative supplier, buy a different lot of a standard you use in your curve, or buy 4 standards and just pick one to use only as a validation….just don’t use your validation standard as part of your calibration curve.

If you just analyze one of your calibration standards again as a sample, you will get the right answer 100% of the time….because somewhere in the software you’ve entered those values in as the answer.  This might look promising, but it tells you ZERO useful information about the accuracy and correctness of your calibration curve.  The validation standard’s “job” is essentially to be an unknown sample, that you secretly know the answer to.

A good basic rule of thumb is that the difference between the known value of your validation standard and the instrument’s reported result based on your curve should vary no more than 10%.  We’ll call this “Validation Recovery” for ease of terminology.  For most applications, 10% is much larger than the recovery should be, but if you’re just started down the path of improving your laboratory accuracy….it’s a good basic place to start.

From here we are going to have to dive into a bit more math, and I think we can all agree that one math topic per post is frankly too much math!

Next week I’ll cover the basic statistics of calculating a more precise validation recovery range for your instrument, tracking validation recovery, and how you can use that information to access the performance of your instrument!

Part 2: What Creates an Accurate Calibration Curve

Welcome back for the second week of Common Calibration Conundrums and Other Laboratory Queries!

 Before we get back into the science fun, I have an exciting announcement.  Bion Sciences now has an Instagram account!  Come follow along with us!  There will be blog notifications, access to our website, and photos of the Bion Sciences team in our natural, laboratory habitat!

@bionsciences605

 Part 2: What Creates an Accurate Calibration Curve

Now that we’ve covered how often you should calibrate an instrument, we should talk about how you pick standards to use for your curve and how many standards you should be using.

Think back to algebra when we first learned the equation for a line.  If your math teacher was anything like my math teacher, they drilled into your brain that 3 points are needed to accurately verify a line and the corresponding slope.  One point is simply a dot in space, and two points allow for too much swing in the correlation of the line, but three points……three points is really where the magic starts to happen!

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Don’t get overwhelmed with the math, I’ll simplify this for you!

The accuracy of your line, and therefore your results, improves dramatically as more data points are added to the line.  Think back to our puppy from last week.  If you only tell the puppy once a day, every day to potty outside, your results might be somewhat….questionable.  Now imagine you tell that puppy 3 times a day, every day, to potty outside.  The chances that puppy is going to catch on greatly improve!  The same goes for your instruments.

When it comes to calibration points, more is always better.  Again, there is no such thing as over calibrating an instrument!

The more data points you provide the instrument, the accuracy of the answers the instrument provides you will also improve.  The only issue that arises from more calibration standards is the time it takes to analyze each standard.  It’s important to find a balance between the most accurate curve you can produce and managing the time restrictions inside your lab.

To deal with time restrictions and busy schedules, sometimes improving your accuracy might involve decreasing the number of calibration points.  (Cue massive shock and awe as I appear to contradict everything I’ve said up to this point! 😊)

Let me give you an example.  Suppose you run 10 calibration standards….but because it takes several hours to build that impressive calibration curve you only calibrate your instrument every other month.  By the end of the second month, how true do you think those 10 data points are….I would argue they probably aren’t accurate anymore.  Instead, what if you ran 5 points once a week, or 3 points every day??  A smaller, more current curve will almost certainly produce more accurate data.  In the world of the laboratory, accuracy is the name of the game!

wyatt

So, you’re staring at a list of calibration standards and you find yourself wondering, “How do I figure out what to order?!”

A good rule of thumb is that your calibration standards should “hug” your expected value.  For example, if your anticipated answer is 3ppm, you wouldn’t want to make a curve using points 0.5ppm, 1.0 ppm, and 1.5 ppm.  You also wouldn’t want to use standards that all have values above your expected answer….meaning that 5.0ppm, 8.5ppm, and 12.25ppm also wouldn’t make a good curve for a value of 3ppm.

For an expected answer is 3ppm, I might select standards with values of 1.0 ppm, 2.5ppm, and 5.0ppm.  Typically, the wider the range of expected answers, the larger the range of standards should be.  Remember, your values need to be hugged by calibration points, not left to fend for themselves on the outskirts of your curve.  Every once in a while you might have a stray value that exceeds the boundaries of your calibration points, and that’s fine….every once in a while!  On the day-to-day and with your typical samples though, your results will be most correct if they are contained within the confines of your calibration data points!

Next week will be, Part 3: How to Know if Your Calibration Curve is Correct.  It’s Big Bang  Theory-themed so be sure to keep your eyes peeled for that!  In the meantime, if you have any questions about this week’s post or any ideas for future posts but sure to leave a comment and let me know!