Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, August 29, 2015

Terminology. Which is it? UPLC, UHPLC or HPLC? The correct name is still HPLC.

Proper terminology is very important in science. I hear and see people misuse the terms "UPLC" and "UHPLC" often so think we need a short post to clarify the correct use of these terms. Here are some basic definitions.

Some quick background information. "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 - 40 bars max). Very large particles are required in this application to aid flow through the support, which in some cases is gravity driven and in others, a small pump is used.

"HPLC" or High Performance Liquid Chromatography: *Used to be called High Pressure Liquid Chromatography to differentiate it from "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. Today, we still refer to all modes of high pressure liquid chromatography separation techniques as "HPLC". The most commonly used HPLC pumps are rated between 400 and 600 bars maximum pressure (with some capable of 1,200 bars or more) 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 where the pump is capable of > 600 bars pressure. UHPLC is still HPLC. Many methods which use sub-2 μm particle columns can and are run on a low dispersion HPLC system 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 HPLC and should be described as such. We have been using narrow bore columns with small supports for more than 30 years and never have changed the name of the technique used each time we changed the column type used (e.g. 20u, 10u, 5u, 3.5u, 2,5u...). 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 HP 1090 HPLC system. Perhaps the technology and methods came too early? Columns proved difficult to pack with the smaller particles (poor RSD). 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).

"UPLCTM" 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 people using the Water's trademark of "UPLC" to describe the technique of HPLC or an HPLC system 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 these cases or even UHPLC, if applicable, not "UPLC" (unless they really are referring to the Waters product technology).

Summary: In general, as long as the back pressure is above ~30 bars and/or you are using packed columns with porous 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 operating at pressures 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" brand system under the same conditions described), but the term HPLC is far more accurate. You are always correct describing the techniques used as HPLC.

Saturday, August 1, 2015

An Often Ignored HPLC & LC/MS Contamination Source. Did you check your Vacuum Degasser?

The introduction of electronic vacuum degassing / degasser modules to the liquid chromatography industry a few decades ago has introduced several new problems which were not seen years ago when we sparged our mobile phase solutions with high purity helium gas. One of these problems relates to how the electronic vacuum degassing modules themselves can contribute to contaminating your HPLC or LC-MS system.

Before using a vacuum degasser module, please review all of the information and advice supplied by the manufacturer of your degassing module. The composition of the degassing tubing has changed a great deal over the past decades resulting in increased degassing efficiency, but also changes in mobile phase compatibility. Some popular solvents may be incompatible with some models in your lab. Make sure you know exactly what types of vacuum chambers and plastics are used in your specific instrument(s). Degassing modules must be operated, cleaned and maintained the same as your other important instruments. When they are not operating properly or are contaminated, they should be serviced as soon as possible or risk further contamination and damage to your system ($$$). 

In a previous post ["Inline HPLC Degassing Modules"] we discussed the convenience that these devices have brought to our laboratories, but also the extra training requirements (such as cleaning and flushing the channels every day) which must be undertaken to use them successfully. When the operational guidelines for the use of these products are ignored, these devices can contribute to the contamination of your HPLC and / or LC-MS system. The internal wettable surfaces of the degassing module contain one or more types of plastic(s) which come into contact with the mobile phase (examples of plastic used: Teflon, Teflon AF and/or Peek are the most common types of used). To effectively remove gas from the mobile phase, the liquid must pass through plastic tubing (or pass by membranes) placed in a vacuum, for a period of time which is long enough to allow the gas to diffuse through the material and out the exhaust port of the degassing module. The degassing tubing (most use tubing) should have the maximum chemical compatibility possible while allowing it to also be porous enough for the gas alone to diffuse through the walls of the tubing under vacuum. These requirements usually result in fluoropolymer tubing (Teflon or Teflon AF) being used as they have broad chemical compatibility plus can be formed with controlled pore sizes for the effective removal of gas, not liquid, through the tubing walls. However, there are exceptions to this and the plastic(s) used may NOT be chemically compatible with all liquids used in chromatography applications. Depending on the plastic degassing tubing used, the tubing may swell, fail or even dissolve into the mobile phase solution! Be sure and check the chemical compatibility chart offered by the degassing module manufacturer for compatibility with your mobile phase and ALL additives used before using the instrument. Some examples of incompatible chemicals on the lists of many instrument vendors include: THF, Chloroform, strong acids or bases, Hexanes and Sodium Azide. Use of incompatible solvents or additives may result in complete failure of the degasser module plus contamination of the entire instrument flow path. *Please do not risk it. Be aware of which chemicals may pose a risk with your system. For example: The use of many fluorinated solvents may dissolve most types of tubing when Teflon AF is used for degassing.
  • Note: We have seen an increase in the use of various perfluorinated solvents, esp with LC/MS systems. This has resulted in severe degasser damage plus MS source contamination (e.g. HFIP and Ethoxynonafluorobutane). Perfluorinated solvents are not compatible with vacuum chambers which contain Teflon AF. They may dissolve the degassing tubing, resulting in the destruction of the degasser and contamination of the mobile phase (IOW: the complete HPLC system). Additionally, we commonly see ion-pairing reagents such as TFA and TBA "sticking" to the plastic used in these modules causing a leaching of material over long periods of time (again, most obvious on an MS system where you can "detect" it in the background signal). These ion-pairing agents must be thoroughly flushed out of the flow path to reduce contaminating the entire system over time. *A strong wash solution with a little acid (formic) alternated with a wash containing some base (ammonium) often helps in this regard. Wash cycles of over 12 hours are often needed to remove these compounds and see improvement. In some cases we must replace some or all of the internal parts of the degassing module to eliminate the contamination. It is for this reason that we should avoid the use of strong ion-pairing reagents in any LC/MS system as they often contribute to very high background signals and long term contamination. *Helium sparging should be considered for such applications.

Reversed phase HPLC applications which use highly aqueous mobile phases may under some circumstances result in high rates of pervaporation of the water vapor into the degasser module resulting in condensation of the water into the vacuum system (Unlike the older Teflon material used, the newer Teflon AF formula is more permeable to water vapor). Once liquid enters the vacuum pump, severe damage has already occurred and failure of the vacuum system soon follows. *If you ever notice liquid exiting through the vac pump's exhaust port, turn off the HPLC system and have the degasser module professionally serviced. 

Another common problem seen when aqueous solutions are used in an in-line vacuum degasser are that of algae and bacterial growth. Most often observed in systems left unused for a period of time or which are not periodically flushed out with organic solvents. Growth inside the low pressure tubing and even inside the vacuum chambers has been known to contaminate the entire flow path of an HPLC system. Replacement of the tubing and internal chambers usually resolves the problem.

Buffers / Additives: Just as with the rest of your HPLC system, any buffer salts or additives which are left in the system (even overnight) can damage it. This is true of the vacuum degasser module too. Please be sure to flush all of the vacuum degassing chambers of any salts or buffers when not in use.

For normal phase applications, high concentrations of n-Hexane may cause contamination or damage to a degasser attached to an HPLC or LC/MS system. Many types of degassers are not compatible with Hexane. The ultra high evaporation rate of hexane coupled to the advanced materials found in the degassing tubing or membranes may result in the hexane condensing on the outside of the internal degassing tubing of the degasser and then aspirated into the vacuum system (causing damage). The contaminants are then transported back through the tubing walls into the solvent stream (your mobile phase).

If your HPLC's vacuum degasser fails to achieve vacuum, has liquid exiting the vacuum pump exhaust port (or exhaust tubing) or shows an error (e.g. Leak Error, Yellow or Red light on an HP/Agilent system or "Degasser Hardware Fault" / "Degasser High Leak Rate" messages often seen on Waters brand systems), then your entire HPLC system may be out-of-compliance - because the degasser is broken. Have the degasser professionally cleaned and repaired so you can put the system back online. Do not assume that only the vacuum pump has failed, as replacement of the pump alone often results in failure of the replacement pump soon after (due to contamination and other problems incorrectly diagnosed). The true cause of the failure must be correctly diagnosed and repaired and this is something best left to professionals to do.

Our professional HPLC degasser repair shop receives many types of degasser modules with leaking or ruptured vacuum chambers. These directly contribute to mobile phase contamination as any seal failure in these normally "dry" systems results in liquid contaminating the vacuum system which in-turn sends contaminated liquid and vapors back into the HPLC mobile phase stream. *Note: If you are using your HPLC degasser with Mass Spec detector, then the resulting mobile phase contamination may contaminate not just your column, but MS source too (costing a great deal of money to decontaminate). Please, at the first sign of trouble, have the degasser professionally diagnosed, cleaned and repaired. For more information on having your degasser professionally diagnosed and repaired with fast turnaround at a fraction of the price charged by most instrument vendors, please refer to this link: "HPLC Degasser Repair Service"