HPLC (high-performance liquid chromatography) and UHPLC (ultra-high-performance liquid chromatography) for researchers are mandatory instruments in their laboratory. HPLC and UHPLC function is to separate, identify, purify, and quantify various compounds. Their users come from many fields, including biomedical research, cosmetics, F&B, environment, and energy.
Both of these instruments work based on chromatographic techniques. Mikhail Tsvet, a Russian botanist, found the chromatography technique in 1903. He invented this technique of separating different plant pigments into a series of colored bands on packed columns. Sixty-six years later, the first HPLC launched. As technology develops, UHPLC appears as an innovation in the liquid chromatography technique. UHPLC offers high efficiency in analysis.
Therefore, the trend of using HPLC is shifting to UHPLC. It is crucial to know your analysis application rather than follow the trend. You must understand how complex your sample analysis is, what analytes are to be measured, how many samples are in a day, what required level of sensitivity, etc.
We’ll discuss the difference between the two and the transfer method from HPLC to UHPLC. Check out our explanation below.
HPLC vs UHPLC
These are the five main differences between HPLC and UHPLC.
- Particle Size
The particle size in the HPLC column/stationary phase ranges from 3-5 µm, while in UHPLC, it is 2 µm or smaller. - Column Dimensions
The HPLC column type has an internal diameter of 4.6 mm and a length of 250 mm. Whilst, the UHPLC column has a smaller diameter of 2.1 mm or less with a shorter column length of, for example, 100 mm. - Flow Rate
The flow rate usually runs on the UHPLC system is lower than the HPLC system. In HPLC, the levels range from 1-2 mL/minute, whereas in UHPLC, it is only 0.2-0.7 mL/minute. - Backpressure
With smaller particle size and column diameter, the backpressure on UHPLC is higher than that on HPLC. HPLC systems usually operate at a maximum pressure of 400-600 bar, while UHPLC can operate up to 1500 bar, as is owned by the Thermo Scientific Vanquish Horizon UHPLC System. - Parameter Detection
Getting a sharp peak shape requires a detector that offsets and provides the necessary number of data points per peak. Most of the detectors are capable of detecting up to 250 Hz. This value is sufficient for HPLC and UHPLC.
In addition, the differentiation between HPLC and UHPLC is their application. HPLC is commonly used in routine analysis, whereas UHPLC uses in research and method development.
Why Choose UHPLC?
The underlying reasons for this include:
- Speed and Throughput
Comes with smaller column and particle sizes, lower system dispersion, and the appropriate pump pressure. Consequently, UHPLC achieves a short time in analyte separation compared to HPLC. One of the examples in this application notes takes 2.5 minutes using a column with dimensions of 4.6 mm. However, using a column with dimensions of 2.1 mm, separation can be achieved in 1.5 minutes. - Better Resolution
Utilize smaller columns and particles to produce better and sharper peaks, thus providing better resolution and peak capacity values. - Lower Costs
The flow rate on UHPLC is lower than HPLC. Besides, the separation process is faster, the solvents used, and the mobile phase will be reduced, resulting in reduced costs incurred.
So, why not all users switch to UHPLC?
Reasons that might be into consideration:
- To achieve higher backpressure and maintain accuracy or precision at lower flow rates, a pump with higher performance is needed, which will add to the cost of the instrument.
- HPLC systems are known more from the beginning than UHPLC. Because of that, almost all methods developed on HPLC. Thus, transferring these methods to UHPLC requires time and resources to optimize.
Can we have both (HPLC and UHPLC)?
You can analyze successfully using both instruments simultaneously with the Thermo Scientific Vanquish Duo UHPLC System in this application note.
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