HPLC Baseline Stabilization Tips for Refractive Index Detectors (RI or RID)

If you use refractive index detection (RID) for your HPLC samples, then you are already familiar with the very long equilibration periods needed to stabilize the system and associated baseline drift. Initial equilibration can take several hours. In fact, re-equilibration takes far longer to achieve with this detection mode than most others (i.e. UV/VIS, FLUOR, EC). While there is no quick cure for these delays, there are a number of things that you may be able to do to minimize or reduce these wait times. Here are a few to consider.

• ROOM TEMPERATURE: Locate the RID in a quiet, stable location. If the room temperature in which your HPLC system with RID system is located fluctuates by even one degree C, that can effect the stabilization of the system. The ideal room to use RID will be away from any windows, drafts, doorways, direct sunlight and HVAC vents or ducts. It should be located in a quiet area away from people walking by it. All of these things can contribute to temperature instability, which is what you want to avoid.

• INSULATION of HPLC CAPILLARY LINES: All of those stainless steel capillary lines leading from your column outlet to the RID's flow cell are loosing heat to the surrounding air (cooling). To reduce this thermal effect, insulate any metal lines with plastic tubing to reduce the heat loss. Most any type of laboratory grade, thick walled plastic tubing can be used. Pass the SS tubing through the plastic insulated tubing or use a section of split-tubing to cover it. Cover as much of the exposed tubing as possible, right up to the fittings. - Note: Sometime the HPLC system's solvent bottles may be subjected to varying temperature changes too. In these cases you can wrap the bottles with an appropriate insulating material to reduce the effects.

• FLOW CELL TEMPERATURE: Modern RID units have a heated flow cell with thermostat to control the temperature of the flow cell. This helps stabilize the temperature inside the flow cell as well as minimize the unintended effect that the heat given off by the RID's electronics has on the temperature inside the flow cell. If the flow cell temperature does not stabilize, then the baseline will drift in response to it. For most methods, select a flow cell temperature which is at least 10 degrees C above ambient (since most of these units can heat only, not cool). Factor in any column temperature used too. If you are maintaining your column at 40C, then try to maintain your flow cell at the same temperature to minimize any differences. Feel free to experiment to find the best temperature for your flow cell. Try different temperatures (in 5 degree C intervals), wait for the system to fully equilibrate, then measure the baseline S/N ratio. You may find best results using different column and flow cell temperatures. Sometimes the room temperature effect can be countered by using an optimized flow cell temperature (higher or lower). Always factor in your mobile phase boiling point (b.p.) into your method and keep the column and flow cell temperatures well below the b.p.

• DEGASSING / DECREASING DISSOLVED OXYGEN: Reduce and stabilize the amount of dissolved gas inside the mobile phase and you may achieve faster equilibration times with a RID. You do not need to remove all the dissolved gas (in fact, a reduction of 50% is often enough). The amount of dissolved gas inside the mobile phase effects the measured refractive index. As it changes, so does your baseline. High percentages of mobile phase dissolved gas = lower RI; Less dissolved gas = higher RI. Now water holds less dissolved gas than non-polar organic solvents (e.g. THF) so this effect is more pronounced when you are running non-aqueous GPC separations, but maintaining a stable dissolved gas level for all mobile phase types is important to reduce baseline drift. Stability is our goal. Continuous degassing of the mobile phase either through sparging with high purity helium gas (best for non-aqueous separations) OR using an inline vacuum degasser should provide you with a way to control the amount of dissolved gas in the solution and reduce drift.

These are a few of the factors which can effect the equilibration and drift times of an HPLC system equipped with a refractive index detector (RID). Careful selection of the instrument module's location, insulating the exposed capillary lines and bottles, optimizing the column and flow cell temperatures, maintaining a steady and controlled temperature in the environment, plus removing dissolved gas from the mobile phase may all contribute to more stable baselines and better quality peak integration. It is also a good idea to review training in the correct operation of the RI detector too. Learning to correctly operate the flush and optional recycle valves on these detectors is critical to their operation. Failure to properly flush the reference cell before each analysis with fresh mobile phase may lead to baseline changes or artifacts.

Another article which may help you improve your analysis method can be found on this site. "Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)".

HPLC Detector Optical SLIT WIDTH Selection

A few notes on HPLC Optical Slit Width selection:

   Notes: 

1. The chosen slit width setting determines the amount of light which is directed to the detector.
2. For most HPLC methods, a slit width value of 4 nm is suggested. 
3. Bandwidth should be set at least as wide as the optical slit width.

Characteristics of Narrow Optical Slit Widths:

• Less light falls on the detector
• Less signal intensity
• Increased baseline noise
• S/N ratio decreases
• Spectral resolution improves which allows for more accurate spectral identification.

Characteristics of Wide Optical Slit Widths:

• More light falls on the detector
• Greater signal intensity
• Decreased baseline noise 
• S/N ratio improves
• Spectral resolution decreases and detail is lost. Less accurate spectral identification and an increase in errors for spectral library matching.

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.

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.