In the last blog post I talked about how a gradient is formed by the two different pumps. Today, we will look at the ability for those pumps to deliver the actual gradient that a scientist requests as well as how precise that delivery is.
Before we do that, let’s take a step back and remind everyone the difference between the words “precision” and “accuracy”.
Am I Precisely Accurate or is it Accurately Precise?
Sometimes we accidentally interchange these words, but there is a very unique distinction that makes it important to differentiate the differences behind the meanings. When we talk about “precision” we’re addressing the ability to do something in a repeatable manner. “Accuracy” deals with the ability to do something and have the outcome be as close to the true value as possible. Figure 1 provides a graphical representation of precision vs. accuracy.
So what is gradient precision and why does it matter? When we talk about gradient precision, the key is to look at an individual system and examine the pump’s ability to deliver the identical composition (even if it is not accurate) from sample to sample. This provides the scientist with confidence that they will obtain an identical chromatographic profile whether it is the first injection or the millionth injection.
When we compare gradient precision for a binary and quaternary, here is the distinction:
When we look at gradient accuracy, a scientist requires that the system delivers the exact same gradient from System A to System B. So when transferring a method from one system to the next, the scientist will have confidence that both systems will deliver the exact same gradient to ensure identical chromatography occurs on each system. This is a different distinction on the behavior of the gradient.
Is Accuracy or Precision Important for Gradients?
In a perfect world, we would have systems that could meet the accuracy and precision requirements without any error.
Let’s look at an example so we can understand what affects gradients and its compositions. Let’s say we want to create a 95:5 (Solvent A:Solvent B) gradient at 1.0 mL/min. We will pick an arbitrary pump head volume of 100 μL, since round numbers are easier to work with.
In this situation, the binary pumps would need to deliver flow rate of 950 μL/min for Solvent A and 50 μL/min for solvent B in order to create the 95:5 desired composition. Several factors can impact gradient precision and accuracy in a binary system. Some of these include (but are not limited to) check valve performance, internal surface of tubing when it is drawn, fittings, compressibility of the solvent, solvent filters, and quality of the solvent.
In the exact same situation, a quaternary pump’s proportioning valve would need to deliver a packet of 95 μL Solvent A and 5 μL Solvent B for each pump cycle. Factors that can impact gradient precision and accuracy in a quaternary system include (but are not limited to) perfect closing of the proportioning valve after delivery of a packet, ability of proportioning valves to open and close fast enough to deliver smaller sized packets, height of solvent bottles relative to each other, and exo- and endothermic effects of the solvents when they are mixed.
With the examples, we see that a lot of factors can affect both pump accuracy and precision, and some of them are out of our control. It is also important to note that “accuracy” and “precision” are intertwined and a system needs both to be able to successfully reproduce methods over the life of the system.
This is also why instrumentation companies advise scientists to maintain their systems and offer qualification services, as these services ensure that the system is working at optimal performance to meet accuracy and precision requirements.
In the next post (chapter 3), I will discuss high pressure vs. low pressure mixing. Stay tuned!
Chapters in this series on LC system operation:
- Gradient formation
- Gradient precision vs. gradient accuracy
- High pressure mixing vs. low pressure mixing (to come)
- How to decide which pump is right for you (to come)