High-performance liquid chromatography is an important analytical tool for a variety of research applications. One of the downfalls of this system is the time it takes to get results. What if we told you there was an HPLC system that generates the accurate results you need without taking all the time?
That’s exactly what UHPLC systems can do for you. But switching to these systems will require some adjustments in your operation. Read on to learn how to switch from HPLC to UHPLC and why you should. More potential problems because of the HPLC column materials are explained in another article.
What Is UHPLC?
Ultra-high-performance liquid chromatography is a variation on the usual approach to liquid chromatography. They make use of increased operating pressures possible, which yields more accurate results in much less time. Higher pressure makes it possible to use particle sizes smaller than 2 µm, which opens a whole new host of possibilities.
Smaller particle sizes mean you can maximize the number of theoretical plates and make your column lengths shorter. You can also expand your optimum range of mobile phase linear velocities. This means you can use those higher velocities without sacrificing separation quality and get the results you need in much less time.
When you’re scaling down in size from an HPLC system to a UHPLC system, you’ll need to adjust the run conditions for the new column. There are some simple calculations you can do to handle this conversion. The first thing you’ll need to calculate is the new column length.
The equation to adjust the column length from HPLC to UHPLC is as follows:
LC2 = (LC1 x dp2) / dp1
In this equation, LC is the column length, and dp is the particle size.
This equation allows you to maintain the same separation in a shorter column length.
Now that you have the column length you’ll need, you can adjust the injection volume. When you decrease the column internal diameter and length, you decrease the overall volume and sample capacity, too. You’ll need to be sure to match the sample solvent to the starting mobile phase composition since the column volume is smaller.
To calculate the new injection volume, VI2, you’re going to multiply the original injection volume, VI1, by the decreased column area. So the equation will read
VI2 = VI1 x [(dC22 x LC2) / (DC12 x LC1)]
If you’re using a larger injection volume than you calculated, check for peak abnormalities and irreproducibility that could result from phase overload.
Flow rate is the volume of mobile phase that travels over time (mL/min) and is the next thing you’ll need to adjust. Linear velocity is the distance that mobile phase will travel over time (cm/min). In order to keep the same linear velocity in a new column, you’ll need to decrease your flow rate proportionally to the decrease in the column internal diameter.
In order to calculate your new flow rate, you’ll multiply your old column flow rate by the square of your column diameter decrease. So your equation is
FC2 = (dC2 / dC1)2 x FC1
in which FC is your column flow rate and dC is your column diameter.
The equation we just discussed is useful for maintaining the same flow rate in a decreased column size, but one of the huge advantages of using UHPLC is they can handle a much higher flow rate. But when you’re working with gradient elution, direct substitution of a higher flow rate isn’t practical. In order to maintain separation under gradient conditions, you have to maintain a comparable relationship among gradient time, mobile phase composition, and column linear velocity and geometry.
Now that everything else has been scaled down for the UHPLC requirements, we need to adjust the time program for gradient elution. This will keep the phase interactions the same.
In order to calculate the new time program, you’re going to multiply the differences in the column flow, the column diameter, and the column length by the old gradient time. So the equation is
tg2 = tg1 x (FC1 / FC2) x (dC22 / dC12) x (LC2 / LC1)
in which tg is gradient time, FC is column flow, dC is column diameter, and LC is column length.
You can substitute increased flow rates into the equation to find a comparable time program if you like. But it’s important to keep in mind the system limitations. Mobile phase changes can be limited by mixing chamber capability, dwell volume, and pump accuracy.
The other thing you’ll need to check when you’re scaling from an HPLC system to a UHPLC system is the detector settings. The data collection rate and time constant must be adapted to the narrower peak shapes. As a general rule, you want each peak to be defined by at least thirty data points.
Depending on the software you use, you may not need to manually adjust the detector settings for the new UHPLC system. Software like the Thermo Scientific Chromeleon Chromatography Data System has a wizard that will take care of calculating the best settings for you. If you don’t have this, check your detector operation manual for further instructions.
Data Collection Rate
As we said earlier, a typical peak requires thirty data points for collection. But when you switch from HPLC to UHPLC, the peak volume and peak width usually decrease. Therefore you must adjust your data collection rate to meet the requisite thirty data points.
To get your new data collection rate, you’re going to divide the old data collection rate, D2, by the square root of the new column length multiplied by the new particle size over the old column length multiplied by the old particle size. The equation will read
D2 = D1 / √ [(L2 x dp2) / L1 x dp1)]
in which D is the data collection rate, L is the column length, and dp is the particle size.
Transfer to UHPLC Today
UHPLC systems can offer some great advantages over traditional HPLC systems. But you will have to do a little footwork to convert your old operations to the new systems. Make sure to account for the capabilities and limitations of your new UHPLC system and you’ll be amazed at the results you can get in a very short time.
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A practical example of method transfer from HPLC to UHPLC is explained in another article.