Terminology. Which is it? "UPLC" (TM) , UHPLC or HPLC? The correct name is still HPLC.

Proper terminology is very important in science. Brand names or trademarks should not be confused with the names of techniques or methods. I sometimes hear and see people misuse the terms "UPLC^TM " and/or "UHPLC" so think we need a short post to clarify the correct use of these terms. Here are some basic definitions of the terms plus background.

"LC" or Liquid Chromatography. A general name for any type of chromatography where liquid is used as a the carrier phase and a solid support is used as the media, often packed into a tube.

 "LPLC" or  Low Pressure Liquid Chromatography. LPLC often includes chromatography analysis using glass or plastic columns with very large particle support beads run at pressures ranging from atmospheric (gravity driven) to several hundred psi (~ 30 bars max, but more commonly just a few bars). Very large particles are required in this application to aid flow through the support, which in some cases is gravity driven, but in others, a small pump is required to push the mobile phase through the column.

"HPLC" or High Performance Liquid Chromatography: *Used to be called High Pressure Liquid Chromatography to differentiate it from the previous term "LPLC". Now we refer to it as "high performance" chromatography, though both terms are technically correct. Compared to the very large media used in LPLC (mm), HPLC uses micron sized support particles packed under very high pressures in stainless steel (note: sometimes strong rigid plastic columns are used for biocompatible applications) columns to enhance the resolution obtained by many orders of magnitude. As such, the more popular definition changed from "High Pressure" to "High Performance" Liquid Chromatography to emphasize this improvement, but once again the technique itself did not change (marketing). Today, we still refer to all modes of high pressure liquid chromatography separation techniques as "HPLC" or sometimes just "LC". The most commonly used HPLC pumps are rated between 400 and 600 bars maximum pressure (with some capable of 1,200+ bars) though in normal use, we rarely run methods which use pressures over 300 bars.

"UHPLC" or Ultra-High Pressure Liquid Chromatography (or Ultra High Performance Liquid Chromatography) has become both a new marketing term and perhaps a subcategory of HPLC (other subcategory examples include: nano-HPLC, narrow-bore HPLC, and mini-bore HPLC). UHPLC is presently defined as the use of sub-2 μm particles with a low dispersion HPLC system, optionally with a pump capable of > 600 bars pressure. UHPLC is still HPLC. Nothing changed except it implies you will use sub 2-micron particles in the column (in other words, just the particle size is highlighted). Many methods which use sub-2 μm particle columns can and are run on a low-dispersion HPLC systems at pressures which do not exceed 400 or 600 bars [For more information, please read: "Pressure Drop Across an HPLC / UHPLC Column"]. The technique used in all cases is still correctly called HPLC and should be described as such in papers, articles etc. 

• For example: We have been using narrow bore columns (2.1 mm ID) containing small particle supports for more than 30 years and never changed the name of the technique used each time we changed the column support type used (e.g. 20u, 10u, 5u, 3.5u, 2,5u, 2.2u...). As a matter of fact, in the late 1980's and early 1990's there was a bug push to use 1.0, 2.1 and 3.5 mm ID columns with 3.5u and smaller particles on low dispersion systems to both save solvent, increase performance and reduce run times. This required the use of HPLC systems which were optimized with low dispersion flow paths such as the Hewlett-Packard model 1090 HPLC system (DR5 pumps). Perhaps the technology and methods came too early? Columns with very small particles proved difficult to pack (poor RSD batch to batch). The solvent savings and reduced run times just did not interest people at that time and after a few years, the lack of interest resulted in few commercial columns being available with these properties (I recall packing many of them myself in the lab).

"UPLC^TM" or Ultra-Performance Liquid Chromatography is a Trademark of the Waters Corporation. Waters Corporation uses it as a marketing term for their own product technology. Defined by Waters as, "the use of sub-2 μm particle columns in combination with low dispersion, high pressure (15,000 psi or 1,034 bar) instrumentation". The confusion seems to come from: (1) New users of HPLC who think that the name "UPLC^" is the name of the technique OR (2) HPLC user unfamiliar with other brands of instruments, using the Water's trademark of "UPLC" to describe the technique of HPLC or an HPLCinstrument with a pump which is capable of exceeding 600 bars pressure OR having a low dispersion flow path. They should be using the term HPLC in all of these cases or optionally, UHPLC, another general marketing term, not "UPLC" (unless they really are referring to a Waters product name or specifically, technology).

Summary: In general, as long as the back pressure is above ~30 bars and/or you are using packed LC columns with particles less than ~ 50 microns in diameter (newer, monolithic supports and superficially porous particles also qualify), then the technique used is always called HPLC. If you are using sub-2μm particles and the system operating pressures for the method are at or above 600 bars, then the term "UHPLC" could be used as well (not UPLC^® unless you are specifically using a Waters Corp "UPLC" branded system under the same conditions described), but the term HPLC is far more accurate. You are always correct describing the techniques used as HPLC and we encourage you do so in all articles, papers and discussions.Key take away ... Changing the particle size of the support used AND/OR operating with system back-pressures above 600 bars does not change the name of the technique used (It is still "HPLC"). Please do not confuse marketing names (created in the hope of selling more systems) with the actual name of the analytical technique.

Common Causes of Baseline Noise in HPLC, UHPLC.

Achieving a flat baseline which does not exhibit spikes, ghost peaks, drift or wander in an unpredictable manner should be a primary goal when performing HPLC analysis or developing methods. Methods which result in flat baselines and have well defined, sharp peaks allow for accurate sample area integration. Integration algorithms perform poorly in quantifying peaks on sloped, drifting or noisy baselines. Excessive baseline noise contributes to many problems, including poor quantitation, high %RSD errors, peak identification errors, retention time variation and many other critical problems. Properly developed HPLC methods are reproducible methods which apply and utilize good chromatography fundamentals. Note: "Noise" is a relative term, often w/o meaning. You should always describe it scientifically, measure and compare the signal to noise ration (S/N) of the baseline vs the peak plus note any cyclical patterns (useful in troubleshooting).

Note: A lack of proper training in the operation of the HPLC system, improper start-up or poor quality maintenance of the chromatograph (Examples: failure to degas and purge the system lines before use; poor mixing; an air bubble stuck in a check valve, a bad detector lamp or a leak will often result in baseline noise) are the main causes of noise. Your HPLC system must be optimized for your specific application. Be sure and allow time for the mobile phase to reach full equilibration with the system before starting any analysis. Do not start an analysis until the baseline is stable.

In this article, we will discuss how temperature fluctuations, inadequate mixing, inadequate degassing and flow cell contamination can result in excessive baseline noise. We will provide suggestions on how to reduce or eliminate these problems. Troubleshooting should be done on-site, not over the web or telephone.

TEMPERATURE FLUCTUATIONS:

To obtain reproducible results, the temperature of the HPLC column must be kept constant during each analysis. Laboratory room temperatures often vary by several degrees during the course of one day and these changes will often change the retention characteristics of the sample(s). The 'On' and 'Off' cycling of power from an air conditioner or heating unit will often cause the baseline to drift in a cyclical manner, up and down, during the day (this can often be seen as a clear sine wave pattern when you zoom-in to study the baseline trace over time). Temperature also changes the refractive index of the mobile phase. Light based detectors (UV/VIS, RI...) will show this change as drift up or down). In some cases, a temperature change of plus or minus one degree C from run-to-run can cause changes in retention times which effect reliability of the method. 

To reduce temperature fluctuations, you must control the temperature of the column and mobile phase (if applicable) during the analysis. This is most commonly done by: (a) using equilibrated mobile phase at the start of the day or analysis, (b) keeping the interconnecting lines as short as possible (esp. any which exit the column and go to detectors/flow cells), (c) insulating any stainless steel lines with plastic tubing to reduce heat loss and (d) using a thermostatted column compartment to maintain the column at a single set temperature throughout the day. Control of the column temperature will remove 'temperature' as a variable from your analysis. Temperature should be a constant run to run, not a variable. Be sure and document the temperature selected as part of your method.

INADEQUATE MOBILE PHASE MIXING:

The associated noise and ripple of incomplete mixing can reduce the limit of detection (LOD) and increase integration error. Both high pressure (with separate pumps) and low pressure pumping (one pump with a multi-channel proportioning valve) systems depend on efficient mixing to reduce noise. For gradient analysis, failure to completely mix the mobile phase solution before it enters the HPLC column often results in excessive baseline noise, spikes and poor reproducibility. "Mixing" is often initially accomplished by combining the flow paths of more than one solvent channel together, using a multi-channel gradient valve or tubing. Mixing also performed directly in a mixer installed in the flow path of an HPLC pump. This mixer is often a static mixer (a simple 'Tee', a tube filled with baffles, a frit or beads, valve orifice or microfluidic device) of low volume design for chromatography use, but allows adequate mixing of the liquids within a prescribed flow rate range. The best mixers incorporate longitudinal and radial mixing in-line. A mixer with too low a volume or of insufficient design can result in poor mixing of the mobile phase (note: incorrect solvent compressibility settings can also cause mixing and noise problems too). To reduce mixing problems, first insure that the mobile phases used are fully soluble with each other. Next, make sure that any mixer used is appropriate for the flow rates and volumes you will be using. If needed, run a gradient valve test to insure that each valve channel is working properly, not leaking or introducing any cross-flow leakage to another channel. Monitor the baseline for pressure stability (% ripple), drift and artifacts  (e.g. spikes) in real time to spot problems and make adjustments to correct them. 

INADEQUATE MOBILE PHASE DEGASSING:

For the best results, continuously degas your mobile phase. Reducing the amount of gas will also improve signal to noise levels of detection, reduce drift and reduce pump cavitation. If you are using an electronic vacuum degassing module, make sure it is maintained and working 100%. A faulty degasser may cause more damage (contamination) to your system and methods. Maintain and Repair them just as you do for your other instrument modules. Gas bubbles may cause check valves to malfunction (get stuck), baseline noise spikes to appear randomly, flow rates and/or pressures to become irregular, detector outputs to show high levels of noise (from air in the flow cell) and also cause the loss of prime or cavitation in pumps. To achieve the best balance of low noise levels and high reliability, both aqueous and organic mobile phases should be fully degassed before and during use. This can be accomplished through stand-alone inline vacuum degassing modules or through gentle continuous helium gas sparging (*Helium makes an excellent choice of gas as it is not soluble in the mobile phase. Never use Nitrogen or Argon gas, they are soluble in the liquid!). In all cases, degassing must be continuous (not just done one time). Continuous degassing reduces cyclical noise and signal variations. For this reason, I do not recommend using ultrasonic baths to degas mobile phase solutions as these are not used in a continuous mode. The mobile phase solution starts to re-absorb gas as soon as you stop sonicating the solution. This results in continuous baseline drift (up and down).

Removal of gasses is critical to the function of a modern HPLC pumping system. The liquids used are compressed to very high levels which forces out solubilized gas from the solutions. This is best accomplished before the liquid is transferred into the pump. These gas bubbles must be minimized to achieve desirable baselines. *Even if you use a high pressure pumping system, an inline degassing system reduces the amount of noise and baseline drift. Properly maintain and service your degasser to insure compliant operation. IOW: Whichever method you use, always degas your mobile phase solutions.

FLOW CELLS:

One other less common cause of baseline spikes and random noise is due to either a dirty flow cell (i.e. the windows) or an air bubble trapped inside the flow cell. If the flow cell is suspected of having one of these problems, then it should be carefully rinsed or flushed out with an appropriate mixture of suitable solutions to expel the air bubble or remove the contamination. If possible, keep a spare, 'known good' flow cell on hand to swap out for troubleshooting purposes. This can help to quickly determine where the problem is. This flow cell must be the exact same size and type (volume and path length) for this purpose. If the cell's windows are contaminated and flushing does not restore them, then many manufacturer's offer kits which allow you to replace the windows and gaskets used. Warning: When attempting to clean or repair any flow cell, be sure and work within the manufacturer's operational specifications for the specific flow cell. Some flow cells are not designed to withstand even very low back pressure and damage can result if you exceed their maximum pressure or chemical rating.

Many other types of problems not mentioned in this short article can also cause baseline noise. For example, a sticking inlet or outlet valve on the pump, worn piston seals, worn out detector lamp(s) or detector electrode (EC) can induce noise. In all cases, the cause must be investigated in a logical, step-wise manner. Demonstrate what is working and rule out items one-by-one.

Reference: http://hplctips.blogspot.com/2014/01/diagnosing-troubleshooting-hplc.html

HPLC PUMP SOLVENT COMPRESSIBILITY VALUES

Have you ever noticed excessive pump ripple (baseline noise) that is not caused by a defective check valve ? The ripple might be due to an incorrect HPLC Pump solvent compressibility setting.

We normally think of liquids as not being compressible in general. Hydraulic systems take advantage of this physical fact and many innovations have been developed using this concept. However, in high pressure liquid chromatography (HPLC) we routinely subject different liquids to very high pressures which can result in measurable liquid compression. The degree of actual compression varies for each liquid (see table). Though the amount of compression is very small, it is enough to change the flow rate of the system. When multiple solvents are mixed together at different proportions, such as is common when running a gradient, the measured flow rate can vary from the set flow rate during the entire run. This flow rate accuracy issue can be compensated for using the built-in solvent compressibility compensation software which is found in most modern HPLC systems. Many of these systems will allow you to manually enter the actual liquid compressibility values for each solvent (pump channel) used. This can result in better baseline stability and less pump noise. I would like to point out that the small improvement gained in performance is best implemented AFTER other major changes have been addressed first (i.e. such as fully degassing your solvents; filtering samples before injecting; selecting the best signal bandwidth and sampling rate values for your detector and insuring that your pumping system has received regular maintenance). 
 
Note how Water has a compressibility value of ~ 46, but a very common solvent such as Methanol has a value of 120. These two are very different. *Most pumps are pre-set with a compressibility value of '100'. A 50/50 mixture of the two run isocratically might benefit from a manually edited compressibility value of 83 [(46 + 120) = 166 / 2 = 83)]. *This is a best guess value as the best compressibility value for a mixture of liquids must be determined through actual experiments. Choose the value which results in the lowest pump pressure ripple and/or noise. 

SOLVENT COMPRESSIBILITY VALUES TABLE:

Solvent	Compressibility (10-6 per bar)
Water	46
Acetone	126
Acetonitrile	96
Benzene	95
Carbon Tetrachloride	106
Chloroform	100
Cyclohexane	113
Dichloromethane	99
Ethanol	112
Ethyl Acetate	113
Heptane	144
Hexane	158
Isopropanol	100
Methanol	120
Tetrahydrofuran	97
Toluene	90

Notes: 
(1) The values shown above are approximate and assumed to be accurate. They were recorded at a temperature of 20C (Reference: Handbook of Chemistry and Physics #90). Various grades/purity of solvent may have different compressibility values so please verify the values of your own solvents before use. These should serve as a general guideline only.

(2) The variation in pressure which occurs between the pump piston compression and decompression strokes are sometimes reported by the pump's electronics to aid in troubleshooting. Agilent/HP brand systems refer to it as the pressure "ripple" (should be less than 0.5 %) and Waters brand systems report the calculated ratio, "Compression / Decompression Ratio" value using this guideline [1.0 - 1.4 = Normal; 1.4 -1.8 = Fair; > 1.8 = Possible Bubble]. In all cases, continously degass all liquids and input the correct compressibility values for each mobile phase solution to achieve the most stable flow.