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.

REFERENCE WAVELENGTHS (as used in HPLC UV/VIS):

One of the most common problems that I see as a consultant in laboratories which use chromatography for sample analysis relates to how to choose appropriate settings for the modern UV/VIS detectors. In addition to selecting scientifically appropriate UV/VIS wavelength(s) and Bandwidth signal values, selecting one optional feature may invalidate an entire HPLC method. This software feature, found in many Multi-Wavelength and Scanning Diode Array UV/VIS detectors (aka: "DAD" or a "PDA") is known as the “ReferenceWavelength” .

• Please do not confuse this specific software feature ("Reference Wavelength") with the initial reference scan ('zero') which the detector takes at the start of the analysis and is subtracted from your desired signal to show only one initial signal plot (and which is used as the initial signal value to compare to the measured signal during the rest of the analysis run. This is usually known as "zeroing" the detector and occurs just once, at the start of each run. When you manually press the 'Auto-zero', you are adjusting the displayed signal plot to a know reference point (often 0.0 volts). This is a one-time zero of the signal and has nothing to do with the special software feature we discuss in this article.

"Reference Wavelength" [Usually written as: Signal Wavelength/Bandwidth: Ref Wavelength/Bandwidth]. Most manufacturers of advanced HPLC UV/VIS (esp. DAD/PDA) detectors provide this extra software feature in their chromatography software, but its use and function are a mystery to most chromatographers. As with all advanced features, proper training is required to understand and use them successfully. Using advanced features without proper training can result in analysis errors, invalid methods and perhaps very expensive product recalls.

Allow me to provide a brief explanation of the “Reference Wavelength” software feature as seen and used with many DAD and/or PDA detectors (e.g. HP/Agilent and Waters brand HPLC systems).

If you are running a gradient analysis, then the change in solvent properties (RI and light absorption/transmission) and temperature over time can cause noticeable baseline drift during the run. This drift up or down relative to the starting baseline reference point is normal, but may cause a number of quantification problems with the analysis reporting software (as flat baselines are more easily and accurately integrated than sloped ones). 

Two scientifically correct methods were developed to deal with this slippery slope of a problem. Each proposed method has some limitations, but if optimized can improve the quality of the resulting baseline (flatter, allowing for better peak integration) and preserve the original acquired signal data for compliance.

(Method # 1) Run the same method again, but this time with no sample (a blank of mobile phase) and subtract the resulting signal to produce a "blank subtracted run". This preserves the original data and removes the observed drift from the resulting signal ('A' - 'B'  = 'C'), but due to the time difference between injections, you are unable to confirm if anything has changed between the time of the first and second injection. It is not perfect.

(Method # 2) Set up the detector to collect a second channel of data (2nd wavelength signal) that is close to the original wavelength selection, BUT far enough away from the original signal such that it will not overlap any of the peak spectra of interest or other compounds in the sample. This is tricky as you want it close enough to show the drift, but far enough away to not show any sample signal. If selected carefully, it can be used as a pseudo blank run for post-run baseline subtraction. You can then subtract the second acquired ‘blank’ signal run from your original signal run and the resulting chromatogram should have a flatter baseline (less drift) for quantification purposes. With this method, two separate signals, 'A' and 'B', are collected at the same time (this is the key). A third, baseline subtracted signal, 'C', can be generated from them. This method preserves the raw data obtained from all three signals (i.e. Original, Secondary, and Subtracted signals). The benefit of this method is that the signals are all acquired using the same time base (unlike Method #1).

Using the concept of Method # 2 described above, many HPLC manufactures added a software feature known as a the ‘Reference Wavelength’ to their systems. This feature allowed a chromatographer to include with each signal choice, 'A', a second wavelength value, 'B', (and bandwidth) as part of the method which would be used to subtract out raw data from the primary wavelength during the analysis. This subtraction occurs in real-time, on your raw data gathered from the detector and the resulting data reported to the user is in fact the result of the subtraction only. The original signal data is destroyed. You will never know what the original data looked like before the reference wavelength was subtracted from it (it has been destroyed). Only the newly manipulated (subtracted) result is provided, 'C'. If any sample peak(s) or impurities appeared in the region where you selected a reference wavelength/bandwidth, then the resulting data would have been subtracted from your actual sample and you would never know it happened or have any record of it! This brings up a serious validation issue as you are modifying the original data with no way of knowing (or documenting) how you have changed it. It is for this reason alone that we teach chromatographers to always turn this feature 'OFF' by default. If they want to make use of the feature, then we suggest that they simultaneously collect data from a second, separate wavelength channel such that the two raw data streams are preserved for validation purposes (Method # 2). IOW: To acquire scientifically useful data, turn 'OFF' the Reference Wavelength software feature and record all of the signal data. The separate signals can be compared, subtracted or manipulated as needed for integration and reporting purposes, but the original signal sample data, 'A', is left unchanged and secure. This allows you to monitor for contamination, impurities, problems or changes during the run. It also allows others to verify your method for accuracy.

Observational Notes:  I am often called in to diagnose what the client's refer to as 'a strange problem' where the area of a known sample peak changes in an unexpected way. That "way" often includes going NEGATIVE, below the baseline. Or even increasing in area, mass or decreasing in mass.The column is clean, pumps work fine, retention times are stable and everything appears to be working fine. *This anomaly is due to the reference wavelength software feature being turned 'ON' and another compound (peak) absorbing in the user selected Reference bandwidth region. Its absorption contributes to the final signal. If the data collected (area) for the 'reference peak' is larger than the sample peak the resulting chromatogram will show a negative peak (this tends to be noticed by most users as it is illogical and indicates a serious problem!), whereas if the reference peak is smaller than the sample peak, the resulting area signal decreases, which may or may not be noticed (incorrectly interpreted as a lower concentration sample). You can see the obvious danger posed by this situation. Companies can be put in a situation where all of their past data is found to be invalid and product recalls may result from this finding. The cause is directly related to a lack of understanding and proper training in the use of the software and/or HPLC system.

 
How to Solve The Problem: The reason we see this feature cause so many problems in laboratories appears to be due to the fact that the Reference Wavelength software feature is being turned 'ON' by default in the software for most DAD/ PDA modules (The real default value for "Reference Wavelength" should always be: 'OFF', not on).  To make matters worse, the default values for the wavelength and bandwidths often supplied by the manufacturers are actually used by most chromatographers (what are the odds that the random values placed in the system are even relevant to your analysis? Why would you use them?). We suggest using a ‘canned’ method template in most laboratories which includes a new default value for this feature... 'OFF' for all analysis methods. Most importantly of all, please obtain formal training in the use of a specialty detector such as a diode-array detector before using one for sample analysis.

Notes

1. The bandwidth chosen for each wavelength is also very important and if chosen poorly, can result in adding noise to your signal, reducing it or even enhancing it. Please refer to this article for more info: http://hplctips.blogspot.com/2011/09/uv-vis-hplc-detector-signal-bandwidth.html 
2. If you are still running HPLC methods with the “Reference Wavelength” turned 'ON' while awaiting approval to turn it 'OFF', then you can ADD additional signals to your method with the same primary settings as before, but with “Reference Wavelength” now set to 'OFF'. Adding the same signal w/o the “Reference Wavelength” will provide you with the original signal data for future comparison to the "collected/modified" signal (allowing you to see if the data was changed). Make sure you configure these extra channels to be saved with the analysis.