HPLC Maintenance & Repair Parts To Have on Hand for HPLC Systems

HPLC (UHPLC) systems are complex instruments which require periodic inspection, cleaning and maintenance. These tasks are critical to maintain the performance, reliability and accuracy of the instrument. If you have not done so already, I strongly recommend that you create formal standard operating procedures (SOP's) which address: (1) The frequency of when routine and non-routine maintenance procedures should be performed; (2) The types of maintenance and/or repair procedures used (e.g. piston seal replacement, A/I rotary valve seal replacement); (3) The exact step-by-step procedure to follow in performing these tasks and (4) The Performance Verification or Qualification steps and procedures which are to be performed to verify that any repairs made have been done correctly. *An instrument log book should be employed to document these procedures over time.

Periodic "General Maintenance" of the HPLC is one type of service procedure which should be scheduled at a set frequency (Example: Every 6 months) and will serve to provide a time to clean, inspect and repair/replace any parts which are worn due to normal use. Such routine HPLC maintenance is often referred to as a basic "Preventative Maintenance" service (or "PM Service"). Spare parts common to your HPLC system(s) should be on hand to perform these scheduled maintenance procedures as part of a normal PM service.

Here is a list of common parts that should be on hand for a "typical" HPLC system used in a pharmaceutical laboratory. Please consult the appropriate manufacture's product literature to determine the correct parts needed for your own HPLC system. This list is presented as a general guideline only:

• Capillary tubing, fittings (nuts and ferrules): Assorted fittings, usually made of 316 Stainless Steel, but could be made of polymeric materials. Always have spare precut and polished chromatography tubing of appropriate I.D. and lengths for use with your HPLC available at all times. Insure that the nuts and ferrules used are appropriate for your brand of HPLC system and the columns used as different manufacturers have different specifications for their fittings and ferrules. Many types are not interchangeable.

• Detector Lamps: At least one spare bulb of a type designed for your specific detector should be on hand. Note that some detectors use multiple lamps so you may need to have more than one type available for each detector. Some lamp bulb types (e.g. tungsten) can be safely stored and last for several years while other types, such as Deuterium bulbs, loose substantial energy after as little as 6 months. If you have several detectors of the exact same design, then there is often no need to stock multiple replacement bulbs for each one. Instead, stock enough bulbs to service one detector as it is unlikely you would see failure of more than one detector on the same day (an exception to this guideline is if you perform PM services on all of the instruments at the same time, then you may want to have multiple bulbs available).

• Pump Pistons: One set of spare new pistons should be kept on hand for each pump module. As with lamp bulbs, if you have several identical pumps, then there is often no need to stock multiple sets of pistons for each one. Stock only as many as you expect to use in one year. Clean and inspect the pistons during each PM for any signs of scratches or surface abrasions. Under routine use, pistons should only require general cleaning and last a long time before replacement is required (> 1 year). Mobile phases which contain high concentrations of salt buffers often accelerate this wear requiring more frequent replacement. *Always install new piston seals when replacing pistons.

• Pump Piston Seals: At least one set of spare new piston seals should be on hand for each pump module. Seals wear out more frequently than pistons. You should go through two or more sets of piston seals before you need to replace the pistons. If the piston seals leak, inspect the pistons for wear (replace with new ones or clean and reuse) and install new piston seals. Mobile phases which contain high concentrations of salt buffers often accelerate this wear.

• Solvent Pickup Filters: These are the large particle filters which sit inside your solvent or mobile phase bottles. They are often made from stainless steel or sintered glass with porous inlets (~10 to 30 micron) and can clog or become fouled over time (esp. when used with aqueous buffers). In some cases these can be cleaned using sonication (not sintered glass filters, only steel or polymeric!). Note: Sometimes it is most cost effective to replace them with new filters then clean and re-use them.

• Inline Frits/Filters: You may have an inline filter placed after your PUMP head, but before the column inlet to collect any remaining particulate matter. These filters can extend the lifetime of the entire HPLC system (esp. the A/S, A/I and Column), but will only do so if changed on a regular basis. Some manufacturers incorporate this type of filter into the design of their pump modules. An example of this can be found on the HP/Agilent brand model 1050, 1100 and/or 1200-series pumps. These have an inexpensive 10 micron PTFE frit installed in the outlet valve of the pump. This filter catches all of the normally occurring piston seal debris and larger mobile phase particles and should be changed every month. Other pre-filters are installed in cartridges just before the column inlet. These often overlooked pre-filters filters must be replaced about once each month to do their job properly. Keep plenty of spare filters on hand.

• Auto-injector Rotary Valve Seals: If you have an auto-injector, then a high pressure valve is probably used to switch the sample into the flow path for analysis. This valve will have one or more parts which require regular inspection, cleaning and periodic replacement. Mobile phases which contain high concentrations of salt buffers often accelerate this wear. The valve rotor seal is the most common part which requires replacement.

• Auto-Sampler Needle: A needle should last a very long time, but depending on the frequency of use and type of vial septa encountered it can require replacement at regular intervals. A good general guideline would be to keep one spare needle on hand for every 2-4 systems.

• Auto-Sampler Needle Seat: The needle seat often requires more frequent replacement than the needle due to repeated mechanical wear. A good general guideline would be to keep one spare needle seat on hand for each system.

• UV/VIS Detector Flow Cell: While not actually a required PM spare part, this one is worthwhile to have. If you employ a UV/VIS flow cell, then I always suggest you keep one dedicated spare flow cell on hand which matches the size and volume of the type you use in your instrument. A spare flow cell can prove to be very valuable as a troubleshooting tool if you believe that you have contaminated or clogged your current flow cell. A quick swap can answer the question and get you back to work quickly saving hours or days of lost time. *Note: This extra flow cell should be kept separate from all instruments for use as a tested spare only and not used for regular analysis.

If you have suggestions for other types of common HPLC spares to add to the list or to have on hand, then please let me know.

UV / VIS, VWD, DAD, PDA HPLC DETECTOR SIGNAL BANDWIDTH (bw) SELECTION

Modern chromatography UV/VIS detectors offer the operator a choice of one to several hundred different signal wavelength choices (as is the case for Diode Array Detectors). Besides being able to specify a single wavelength, you can often choose a signal BANDWIDTH (bw) to associate with each wavelength [e.g. for a 280 nm signal with 10 nm bandwidth. This is often written as: 280 (10) or [280:10]. In many detectors, Signal Bandwidth is a variable, not fixed and represents the total number of nanometers across the specified signal value chosen. For example: If you select a signal wavelength of 280 nm and choose a bandwidth value of 10 nm, then you are actually gathering all signal data between 275 nm and 285 nm (5 nm to the left of the apex and 5 nm to the right for a total of 10 nm). Using a narrow bandwidth has the advantage of increasing the signal selectivity of the detector as you are only collecting data within a tight window. If you were to increase the bandwidth to 60 nm in the same example you would now be collecting data between 250 nm and 310 nm. The additional data collected over this wider range may reduce the total noise (by averaging it over a wide range), improve the S/N ratio (which may increase sensitivity), but it also reduces the selectivity. Large bandwidths also increase the chance you may include peak signal data from other co-eluting components into your signal data. You must select a bandwidth range for each signal wavelength which is located 'safely' away from any other potentially interfering peak. As with many things in life, balance is important. In this case, bandwidth choice is the balance between selectivity and sensitivity.

• When developing new methods we recommend that you choose an initial bandwidth value of 10 nm for each signal. This provides a nice balance between selectivity and sensitivity. It is also a common bandwidth value used on many older UV/VIS detectors which have a fixed signal bandwidth (such as many single or variable wavelength detectors).

• If you have determined the exact signal maximum for your sample and you would like to gain additional sensitivity for your sample (and thus decrease selectivity), re-run the analysis using several different, but increasing signal bandwidth values (e.g. 10, 20, 30, 50 and 100 nm). Choose bw values that are safely within the range of the detector, within the limits of the mobile phase's absorption region and also away from any potential co-eluting peaks. *To confirm which value is best, be sure and calculate the actual measured signal to noise ratio of the peak of interest after each analysis. This is a critical step! Do not be fooled by increases in the peak height or area alone as these changes are not always synonymous with better signal to noise ratios. Only by measuring the actual baseline noise level for each run and comparing it with the actual peak signal obtained will you be able to determine if increasing the bandwidth has provided you with better noise reduction and signal strength.

• To increase spectral signal selectivity choose a bw value that is very narrow. A value such as 2 or 4 nm would allow the detector to collect only signal data that is at or near the apex of your selected wavelength. This can be very useful when trying to discriminate your signal from nearby signal peaks, especially at low wavelengths such as 210 nm.

• When reporting your method conditions always include the wavelength AND bandwidth used for each signal. In order to accurately reproduce your method, this information is needed. *The flow cell dimensions, wavelength and bandwidth should always be included in your method.

HPLC Flow Cell Volume & Path Length:

Modern UV/VIS detectors offer several different flow cell options. The option(s) you select can make a big difference in the level of signal sensitivity, sample dispersion and response you obtain. If you fail to note which type of HPLC flow cell you use in a particular system, then you may discover some problems when transferring a method to a different instrument. Always record the flow cell volume and path length used as part of your method description. 

Flow Cells Usually Differ In Three Ways:

(1) Maximum Rated Back-pressure;

(2) Flow Cell Volume and

(3) Flow Cell Path length. 

Let’s take a look at these in more detail.

• Maximum Rated Back-pressure: Unless the detector is in series with another detector, column or has a back-pressure regulator on it, the expected back-pressure on a typical flow cell’s outlet is just about one bar as it usually is directed to an open waste line. *This topic will be discussed in more detail in the future as part of another “hint and tip” topic. Today we are more concerned about the remaining two options:

• Flow Cell Volume: Analytical flow cells are commonly offered in nl to ul sizes. Depending on your instrument setup, column and sample(s), one flow cell volume may make more sense than another. After you have spent time separating and concentrating the peak of interest into a tiny volume you do not want to elute it off the column and mix it with another peak because the cell volume is too large. Ideal cell volume is a compromise between sample dispersion and sensitivity. The best choice will be determined mostly by the actual peak volume of your separated sample. The general rule is that your flow cell volume should be no larger than 10% of your peak volume and ideally ~ 2.5% (a 1:40 ratio), but there are some exceptions to this rule. When in doubt, experiment with different cells and do not forget to consider the total volume of all the connecting tubing and valves in your system as these contribute to many issues when the column volume decreases (such as when using mini or narrow bore columns are used). Some common analytical cell volumes offered by various manufacturers are 2 ul, 6 ul and 13 ul. For narrow bore columns (~ 2.1mm ID) a smaller cell volume (~ 2 ul) will result in less sample dispersion, while a larger cell volume may increase overall sensitivity (esp. when used with a longer path length). Mid-bore or Mid-Size columns (2.1 to 4.6mm ID) often are best suited to cell volumes around 6 ul to minimize dispersion and still provide good sensitivity. Larger flow cells such as the common 13ul size often have longer path lengths which can be used to enhance sensitivity. Standard 4.6mm ID columns often benefit from a 13ul volume cell to provide maximum sensitivity with less concern for dispersion effects when larger columns are used (e.g. 4.6 x 250mm). Keep in mind that these are general guidelines only. Most samples contain many peaks of varying width & volume, so you will need to select the cell volume that is optimized to most of the peaks found in your sample.

• Flow Cell Path Length: The flow cell’s path length affects the intensity of light reaching the detector (Beer-Lambert law). For the same volume of sample, the apparent concentration of the sample will appear to be higher if the path length is longer. There is no established standard for ‘path length’ so it is important that you always known what the path length of each flow cell is in your detector (10 mm is very common). Just as volumes vary, manufacturer’s offer different flow cells with varying path lengths. Even identical detectors can use flow cells with identical volumes, but have different path lengths. When comparing the analysis results obtained from two different instruments, always make note of the flow cell dimensions used in each instrument. If the method is to be accurately reproduced on a second system, then the flow cells used should have the same geometry (volume and path length). One way that the difference in path length can be used to enhance sensitivity of an existing method is to use a flow cell with a longer optical path length. For example, if your current flow cell has a path length of 6 mm you could replace it with one having a longer path length of 10 mm. This would increase the sample peak response (as more light would be absorbed) in your method. *This fact can be useful to squeeze out additional sensitivity in a method and often does not require any change of column or conditions.