Determining the Data Acquisition Rate (Sampling Rate) For Your HPLC Detector

Another common question I am asked is how to set-up the HPLC detector’s sampling rate. This article is specific to commonly used UV/VIS, not mass selective detectors (Mass Spectrometer detectors are set-up in a similar manner, but you also want to take into account the numbers of MRM transitions for each peak and dwell time to account for the scanning delay. Typical values for MS are >10 points with 15-20 being best). 

Most HPLC (UHPLC) instrument manufacturer’s provide default sampling rate values within their software packages. Please do not use them as the values shown were just put there to fill in the data field and may not apply to your application or method. Many chromatographer's use these values without first understanding if they are appropriate for their own methods. This is a common mistake. Just as the manufacturer does not know what wavelength, flow rate or mobile phase you will use, they also do not know what sample(s), method and/or conditions are appropriate for your specific application. As such, they provide numerous default values in these data entry fields to satisfy the software's requirement. Just as you select an appropriate wavelength and bandwidth, you should always calculate and enter the correct detector data acquisition rate value yourself which is appropriate for your specific application, detector type and method. 

The Peak shape's role during integration: For each chromatographic analysis you must determine the optimum sampling rate for the chosen detector. An accurate value is critical for proper instrument set-up, quantification and integration of your sample(s) peaks. In the most basic sense, the area under a perfectly Gaussian peak requires at least ten points to describe it with some detail. Ten points will provide basic data about the shape of an ideal peak to the computer. Since peaks are rarely perfectly symmetrical, a larger number of points will provide more accurate integration of the peak’s actual shape and total area. This will improve run-to-run reproducibility and quantification. We suggest you include twenty to thirty data points to allow for a more detailed fit to the peak. Too few points across a peak and you lose detail and sacrifice reproducibility. Too many points and you start to introduce noise into the system. 


With these facts in mind we can next think about calculating the detector’s data acquisition rate. You must select a data rate (sampling rate) that is sure to provide the recommended 20 to 30 data points across the peak width (we use the commonly calculated peak width at half height as the time measurement). Select a detector sampling rate that will provide you with this degree of detail and resolution. This is best accomplished by initially looking at an actual chromatogram of your sample. Look at the chromatogram and use the narrowest sample or standard peak past the void time, with good retention as an example to determine the best acquisition rate. The narrowest peak will be the worst-case scenario and will insure that you have enough points across all of the remaining peaks in the sample. It's width is often measured in units of time (seconds/minutes). This data can often be read directly off of a generated data acquisition report.

Examples:

(a) If your narrowest peak has a peak width of 1.00 minute (60 seconds), then divide 30 points into 60 seconds for a result of 2 seconds per data point. The preferred sampling rate would be 2 seconds, 0.03 minutes or 0.5 Hz (depending on the units used by your detector).

(b) If your narrowest peak has a peak width of 0.20 minutes (12 seconds), then divide 30 points into 12 seconds  for a result of 0.4 seconds per data point. This equals a sampling rate of 2.5 samples per second or 2.5 Hz.

Summary:  

     To Determine the Data Acquisition Rate For Your Detector You Need To:

• Calculate the best data rate for each method and not use a generalized value (though similar methods will often use the same rate).
• Use your existing sample integration data results to identify the narrowest chromatographic peak in your analysis (at the baseline or half-height).
• Record the width value of this peak (usually in units of time).
• Divide this number by thirty (30) to determine the preferred sampling rate.
• Use this value, or a value close to it, for your detector’s sampling rate.

HPLC Capillary Tubing Connection Volumes:

The length and internal diameter of the HPLC interconnecting tubing used in your system really does matter. The total volume contained in the tubing can dilute your sample or separated peaks. This can effectively undue the work of separating the peak(s) on a column. Extra volume in the tubing can also have the effect of increasing the gradient delay factor for your method (the greater the volume of the tubing from the pump head to the column inlet, the greater the delay in the solvent mixture arriving at the column). In general, keep the the total delay volume as low as possible. This is accomplished by connecting the various modules together using the shortest lengths of tubing possible. For systems which use standard sized HPLC columns (e.g. I.D.'s of 3.0 to 4.6mm and lengths from 100mm to 300mm) the tubing internal diameter should be 0.17mm (0.007"). For systems which use very short, mini or micro bore sized HPLC columns (e.g. I.D.'s of 1.0 to 2.1 mm and lengths from 50mm to 250mm) the tubing internal diameter should be 0.12mm (0.005"). Looked at another way, if the total column volume is less than 750 ul, consider using the smaller internal diameter tubing (0.17mm) to reduce band broadening. 

Here are some tubing volumes to help you evaluate the effect changing the I.D. or length has on the tubing that you use.

I.D. (mm)	I.D. (inches)		ul / cm	ul / inch
0.12	0.005		0.127	0.323
0.17	0.007		0.249	0.632
0.25	0.010		0.507	1.288
0.51	0.020		2.026	5.146
1.02	0.040		8.103	20.581

HPLC Mobile Phase Filtering & Solvent Inlet Filters

HPLC Mobile Phase Filtering: 



The tubing and valve passageways of the HPLC system are very narrow and clogs can result from using solutions which have not been properly filtered. Columns are expensive and will also clog up with particulate matter causing increased back pressure and/or changes in retention times. Running clean, particulate free HPLC grade solvents through your chromatograph is a basic maintenance requirement. High grade chromatography solvents (and ultra pure water) are often pre-filtered through 0.2 micron filters by the manufacturer to meet their grade for use in chromatographic systems. However, there are times when you also prepare (mix) your own mobile phases using theses solvents with or without chemical reagents and additives. When you prepare mobile phase using these reagent grade chemicals or additives you should also take the extra time to filter the final mixture through a 0.2 micron glass or steel filter prior to use. This helps to insure that you start with as clean a solution as possible. *This is a critical procedure to follow with buffer solutions. When using aqueous solutions, possible bacterial and algae growth can occur so remember to date the solutions and dispose of them after a suitable time period (Make up only what you will use in one week). Do not re-filter these solutions and then use them again.


HPLC Solvent Inlet Filters:

Most HPLC manufacturer's supply solvent inlet filters on the lines which draw solvent into the pump head. To protect the pump and components downstream, these lines often incorporate a filter. These solvent pre-filters are usually made from plastic (PEEK or PEAK), glass or stainless steel. Their porosity is typically ten or twenty microns. A smaller porosity could be used, but it would restrict the lines ability to draw up fresh solvent into the pump head at the required flow rate so a compromise in pore size is necessary. The filter is primarily designed to stop the pump from drawing up any large particles or debris which could cause damage to the system and is NOT used to filter the solution (as mentioned above, the solutions used should be pre-filtered). These filters can clog up over time and so should be monitored for restrictions. Stainless steel filters can be cleaned using sonication and heat. Plastic filters should usually be replaced with new ones. Glass filters, which are often made of sintered glass, can be washed, but should never be sonicated to clean them as this can cause the glass to fracture and plug them up even worse. When in doubt, replace them with new filters. Filters used with clean organic solvents often last for many years. Filters which are used with aqueous solutions last for shorter times due to build up of undesirable biological matter.

• Another way in which you can insure a clean source of liquid for your HPLC system is to make sure that your mobile phase reservoir bottles are clean and free of dirt and dust during use. Keep them covered. Always wipe off any dust and debris from the solvent bottles before you uncap them and pour them into another container (much of the dust in the mobile phase comes from dirt that falls into the bottles). Instead of 'topping-off' bottles, replace them with clean bottles containing new solution.

HPLC PUMP SEAL WASH & FLUSHING THE HPLC

Many instrument vendors offer an HPLC Pump with "Piston Seal Wash" option. If you often operate your instrument with high concentrations of aqueous salt buffers (e.g. Protein, Peptide Separations), then an optional seal wash system might be something to include on your HPLC system. When combined with daily flushing of the HPLC system to remove buffers, a piston seal wash system can extend the life of and/or reduce the maintenance needed on your HPLC system. 

NOTE: If your HPLC system has a piston seal wash feature installed, then failure to utilize it on a regular basis (leaving it "dry"), may result in decreased lifetime of the pistons, wash and piston seals, plus leaks due to the added friction. If you have a piston seal wash system, but do not need it (i.e. running only NP solvents), then replace it with a non-seal wash system (manufacturers offer kits) or begin utilizing the piston seal wash feature to prevent damage.

To prevent the build up of buffer salt crystals inside of the narrow bore tubing, LC pump and other HPLC components we strongly recommend that you wash the system down each day, after use. We routinely see HPLC systems with large amounts of white fluffy crystals built up around the pump heads, pistons and various fittings from lack of daily maintenance. High concentrations of mobile phase containing salt buffers in your system (e.g. 0.1 M is considered 'high', but all buffers should be flushed out) can damage the pump's pistons, pump seals, injector parts and are corrosive to the stainless steel used. The resulting damage can lead to expensive repairs and lost time.

• Two types of flushing techniques can be employed to reduce the damage caused by these salt buffers and extend the life of the HPLC system. Flushing the entire HPLC flow path with a solution which does not contain any buffers ('water' to rinse it) and optionally, flushing the back side of the pump pistons using a piston "seal wash" system. Let us consider these two systems.
  

(1) Flushing the HPLC Flow Path: Potential damage from salts can be avoided if you remember to always flush down the entire flow path of your HPLC each day with a proper mixture of HPLC grade WATER plus some organic solvent (to prevent the growth of bacteria and/or mold). Flush the column down first with an appropriate solution to remove any buffers and then remove it from the flow path. Next flush the entire HPLC system down to rinse it of any remaining deposits (sometimes the column can be left in-line and flushed with the system. Consult with  the column manufacturer for advice). The exact flushing mixture to use will depend on the exact type of mobile phase you are using, but pure water is often a good initial choice. You want to select a solution which will dissolve ALL of the buffer used in your mobile phase back into the solution plus incorporate some organic solvent component to reduce the surface tension and also deter the growth of bacteria over time. For example: A common Reverse Phase (RP) wash solution of 80% HPLC Grade water and 20% Methanol can be used in many applications. If you have an automated HPLC system, then this entire process can be stored as a  "RP System Flush" method and programmed to run at the end of each day's sequence or series of runs so you do not have to remember to do it manually.

(2) Piston "Seal Wash": When running with buffers, the HPLC pump's pistons are coated with buffer solution. Over time, the liquid evaporates leaving a film of buffer salts deposited on the pistons. These salts accumulate and can scratch the piston surface or get stuck in the piston seals allowing air to enter the piston chamber and/or leaks to occur (drips from behind the piston seal). Early replacement of the pump head's piston seals and pistons often results from this damage. Washing the internal flow path of the HPLC system (as described in section #1 above) does not wash away all of these salt deposits. A piston"seal wash" system can be used to help wash the back-side of the piston washing away remaining deposits stuck to the piston. The piston seal wash pump's inlet line can be placed in a bottle containing fresh wash solution and through either an automatic timer feature set in the pump's software or through the operator manually turning the wash pump on and off (some systems just use gravity), it can wash the back of the piston area to rinse these deposits away. The rinse solution used to wash the pistons will depend on the type of mobile phase you are using (just like the HPLC flushing solvent). For most RP applications, I recommend a mixture of HPLC Grade Water and Methanol (50/50 to 80/20). Other common seal wash solutions might include: 80% HPLC Grade water and 20% IPA or 80% HPLC Grade water and 20% ACN. For most applications, I prefer using Methanol over IPA because it is much better at dissolving many of the buffers used. A third option would be to use a wash solvent which is the same as your mobile phase, but without any buffers added (try to include at least 20% organic content). Before starting, you must review your own methods to determine which general system wash and piston wash solution(s) are best as their is no such thing as a 'universal' wash solution that can be used with all methods.

If you are using Normal Phase (NP) applications, then the piston seal wash system can also be employed to keep the pistons 'wet' during operation and avoid excessive wear and noise (and that high pitched piston squeal noise), which are common when running dry solvents (e.g. Hexane). Manufacturers often provide special piston seals designed for use with normal phase solvents, but sometime the incorporation of the mobile phase as a seal wash solvent can help lubricate the pistons too. IPA can often be employed as a NP piston seal wash solvent as it is one of the best solutions to use in maintaining the seal over time (IPA is an excellent seal wash solvent for many NP applications). In any case, always make sure that the tubing used in your seal wash pumps is chemically compatible with the wash solution you choose.

• Piston Seal Wash SEALS: One final note about HPLC systems which use a "Seal Wash" system. Some designs (not all) incorporate a separate piston seal, behind the main pump head seal, to seal the rinse solution inside the wash area. Just like the piston seals at the front of the pump head, these wash seals require regular replacement. If your HPLC system uses a wash seal, be sure and have some extras on hand so they can be replaced when you service the pump head. Failure to replace these worn seals usually results in liquid leaking out the back of the pump head. This may be mistaken for a seal failure at the front of the pump head, so you need to be aware of their use to diagnose and repair any leaks correctly.