Method Development Hint: Use your HPLC Diode Array Detector (DAD or PDA) as a Spectrophotometer

One of the many useful features of a UV/VIS scanning diode array detector is that it can be employed in flow injection mode to scan a sample and provide you with some useful data about the absorbance characteristics of the sample (which probably contains a mixture of components). Unlike a spectrophotometer, you only need about 1 ul of sample instead of a 1ml cuvette and only 15-20 seconds of time to gather the data.

Why do this? I use this feature often when I receive a new and unfamiliar sample for method development. I set up the detector to scan and store all wavelengths, in steps of 2nm, from 210nm to 450nm and inject the sample in flow injection mode (that means no-column is present and I easily do this using the By-Pass position on my column selector). In a very short amount of time I can view the resulting spectra of the sample which aids me in selecting the initial discreet wavelengths to monitor. For example: If I notice that the sample shows some absorbance at 410nm using the flow injection run, then notice while developing the analysis method that none of the peaks seen show absorbance near 410nm, then I can assume that I may still have some components retained on the column.

Setup Hints:
(1) For this to work well, you should have a high performance, low volume switching valve or automated column selection system (e.g. The LC Spiderling Column Selection System) installed so you can easily by-pass your column (otherwise, remove your column and place a high pressure, low volume union in its place).
(2) Set the diode array detector to a high sampling rate because the sample is going to fly through the flow cell quickly. Use a sampling rate that is faster than you would use if a column was there to disperse the sample and slow down the peaks.
(3) Choose a wide range of wavelengths to scan and store. If the sample appears colorless to the eye in solution and I am running in a UV transparent solvent such as acetonitrile, then I often use a range of 210 to 450nm.

Proper Wavelength Selection for HPLC Method Development (or Purity Determination)

Selecting the best HPLC wavelength(s) to monitor during an analysis method for use in quantitation and/or purity determination requires both knowledge and careful attention. Here is the basic procedure to use:

Step 1. Create the Method. To determine which UV/VIS detector wavelength(s) should be chosen for the analysis of your sample, you will first need to create a general HPLC Method which retains and resolves the compound(s) of interest on the column (goal is a K prime of >2.0, less than 10.0). Be sure and utilize a scanning diode array detector in full scan mode (often referred to as a photo-diode array detector, PDA or DAD) to scan all relevant wavelengths of your samples (e.g. 210 to 450nm). Note: Your choice of mobile phase and detector settings will effect the S/N values.

Step 2. Determine the lambda max of the sample's spectra using the Data analysis software. Once you have completed the analysis, review the spectral data to determine which prominent peak wavelengths have the maximum signal to noise (S/N) ratio. These “peaks” can be used as the individual wavelengths for integration and purity determination. By sure and double check that any detector options which use a “reference wavelength" are turned ‘OFF’ when running these methods (more info on “reference wavelengths” can be found on this blog in another post). With the wavelength selected, chose an appropriate bandwidth for use (narrow).

Step 3. Edit the HPLC method to use the discreet wavelengths found in step 2. Whenever you run a real sample, continue to use the full scanning mode of the detector so you will know about any other components which absorb at wavelengths far away from and/or near the peak wavelengths. These compounds can add or subtract signal from the main peak making it appear to be more or less concentrated (or more or less pure) than it actually is. If you only monitored the sample with a single wavelength detector, then you would miss this vital information and make errors in your purity or concentration determinations.

Conclusion. (1) Using a multi-wavelength, scanning HPLC detector such as a DAD is one of the most important tools you can use to create accurate and reliable chromatography methods. Always use a scanning DAD for method development to prevent errors. (2) Learning how to correctly use and set up the detector's settings, parameters, special features and options may prevent false or misleading results. Only after you have developed a reliable and repeatable method with good sample retention and peak shape can you begin to report accurate integration and concentration values (and/or make UV/VIS "purity" determinations).

Troubleshooting HPLC Injectors (Manual and Automated)

Sample injectors are a critical component of a chromatography system. Understanding how they operate as well as the proper techniques to use and maintain them are fundamental skills needed to operated an HPLC system. Lets briefly discuss some of these fundamentals as applied to a standard manually operated HPLC injection valve and also in an automated mode as found in an autosampler. *Note: You should always refer to the specific manufacturer's product specifications, operation, servicing documentation or support personnel before servicing any injector.

MANUAL INJECTION VALVE Notes:

These valves allow you to use a high precision syringe to manually fill a fitted "loop" with a sample and then, by turning a valve handle, introduce the sample to the high pressure flow stream directed toward the column inlet. Sample loops are available in a wide range of volumes and take just minutes to install. The injector valve has very tiny openings inside which are moved between two different positions (LOAD and INJECT). The LOAD positions allow the valve to seal off the internal high pressure flow from the loop to allow it to be safely filled with sample at atmospheric pressure. The INJECT position introduces the liquid contained inside the loop to the main flow path (under high pressure). The parts must be clean and seal well to insure proper function. Leaks from all areas (except the vent) are not acceptable and indicate a problem. Here are a few tips regarding the use of manual injectors in HPLC.

• Use the correct type of sample syringe. Usually these are high precision glass syringes with Teflon plungers. The needle tip style is the most critical item! Most injectors are designed to only work with a needle which has a squared off tip (NOT a point as is commonly used in GC!). The most common gauge used is #22. Always check with the valve manufacturer to determine the correct style and gauge of needle before use.
• Leave the valve in the INJECT position during the entire run to flush it clean of sample and stay equilibrated with your method. Switch it back to LOAD only when you are ready to load a new sample.
• For high reproducibility and accuracy within one HPLC system, fill the loop with at least three times the volume of the loop with sample to insure that the entire path is full of sample. This is known as the complete or over-filled loop method. *Choose your loop volume size with this in mind. Loading the same volume as the loop will often result in poor accuracy.
• Loops often do not contain the exact volume stated on them. They can be off by ~25% so consider this when injecting partial volumes and not using the standard over-filled loop method.
• Types of common leaks: (1) Leaks at the needle port (needle seal worn); (2) Leaks behind the valve stator (worn rotor seal, buffer crystals dried inside, over pressured, scratch on rotor); (3) Leaks at the vent (liquid should be expelled from the vent when filling the loop only. Other leaks indicate a problem). Note: Rotor seal damage can cause sample carry-over problems so valves should be inspected at regular intervals (~ 6 months).

AUTOMATED INJECTION VALVE (auto-injectors) Notes:

These valves use a high precision syringe or high pressure pump to fill a fitted "loop" with a sample and introduce the sample to the high pressure flow stream, all automatically. Most function exactly the same as described above, though some are based on true high performance liquid chromatography pumps so have no "syringe" at all (e.g. Agilent 1100/1200 designs) Here are a few tips regarding the use of automated injectors in HPLC.

• Regular maintenance is even more critical with auto-injectors since you often can not see what is going on during the injection cycle. Leaks, if present can be harder to find so make it a habit to visually check all of the areas around the injector regularly.
• Needle and Needle Seats are normal wear items on these instruments. As such, they require routine checking for leaks or damage and replacement when worn.
• Vial Caps: If you make multiple injections from one vial (or large volume injections) with a tightly sealed vial cap, a vacuum can form inside the vial causing volumetric errors to occur in your samples (resulting in you injecting less sample each time). Leave the caps slightly loose to avoid this problem. Multiple injections into the same vial can also cause the needle hole to become enlarge over time allowing the sample or solvent to evaporate over time, changing the concentration of the sample (more concentrated). Replace the cap and seal with a new one if used multiple times. Always leave the cap slightly loose.
• Loop volume: Autoinjectors often incorporate one large loop to handle a wide range of sample volumes. This is a trade off of accuracy for convienence. Accuracy is often poorest at the very low end of the range and best near the middle to high end. Always verify the reproducibility of the injector to inject a specific volume through statistical analysis of repetitive injections.
• Types of common leaks: (1) Leaks at the needle seat (needle seat worn); (2) Leaks behind the valve stator (worn rotor seal, buffer crystals dried inside, over pressured, scratch on rotor); (3) Leaks at the vent (liquid should be expelled from the vent when filling the loop only. Other leaks indicate a problem). Note: Rotor seal damage can cause sample carry-over problems so valves should be inspected at regular intervals (~ 6 months).

These are just a few tips related to HPLC injectors. Please consult the service documentation for your specific instrument to better understand how the system works and what areas you should be monitoring. Understanding HOW these systems operate (and can fail) is one of the most important skills you can learn as a chromatographer. Take the time to understand the complete flow path of your system before using it.

HPLC Solution Degassing, Sparging With the Wrong Gas (Gas Choice Matters)

The other day I took a call from a client whom explained they were having a number of problems with their HPLC pump. They felt that they were very experienced chromatographers whom had been unable to find the reason for why their pump flow stability was poor. It had very high ripple and noise. The pump had been fully serviced one month earlier and passed all qualification tests. Their UV/VIS detector appeared to work fine and was ruled out as being the problem early on. They used HPLC grade filtered solvents, operated at an appropriate flow rate, had a clean and tested column installed, always primed their pump before use each day and sparged each solvent reservoir with a low stream of continuous gas kept away from the solvent inlet lines. Everything seemed in order, but something was clearly wrong. Their vendor suspected the check valves were to blame so they purchased and installed new ones with no change. They still had an unstable flow rate under all conditions tested (pump pulsation of 5%). When I asked them if they had changed anything related to the HPLC system in the past few months I was reassured that nothing had been altered.


Often the best way to solve a problem is to start at the beginning. Take nothing for granted. This started as one of those many phone calls I receive where someone wants me to solve their problem over the phone and by not visiting their laboratory. Sometimes this is possible, but sometimes the problem is something that can only be seen by being physically present in their laboratory. I felt this was one of those times. So, they agreed to pay for a few hours of consulting time to have me come out and go over their system to find the problem. Once I arrived at the client's lab I quickly went over to inspect the layout of the equipment and check the tubing connections for the correct fittings and tightness. Next, I looked at the software parameters being used to operate the system. Some small issues were found, but not enough to explain the problem seen. I then looked at the physical output of the pump and detector to get a better idea of the period, cycle and type of noise seen. While I was reviewing the data and still looking over the system, I found the problem. The high pressure gas cylinder next to the instrument was labeled ARGON. Argon was being used as the sparging gas for the mobile phase instead of the more appropriate gas, Helium. They had in fact recently switched to argon gas because it was less expensive to use than helium. The person (their senior chemist) who had made this substitution was rewarded for his cost-cutting suggestion. Their choice of argon gas had of course cost them several weeks of down time while they tried to solve this problem on their own.... not much of a savings when you consider that! So, they had in fact caused the problem themselves, but were not aware of the fundamental reason why changing to argon gas was a very bad idea.

Why does the gas choice matter? For liquid chromatography applications we only use high-purity helium gas for sparging because it is one of the few inert gases which is the least soluble in water and mobile phase solutions. Gases such as argon and nitrogen ARE soluble in water and mobile phase solutions.  While they can be used to displace oxygen from air (great if you are making wine, but not so great if you are using the solution for HPLC), they infuse the liquid with gas (like a soda). Helium easily displaces air (oxygen and nitrogen) from solutions while not adding significant amount of dissolved gas to the solution. Helium is the least soluble and most inert gas to use. If we sparge with argon or nitrogen, then we infuse the solution with gas. This is the opposite of what we wish to accomplish by degassing our mobile phase. Please, if you wish to use the continuous gas sparging method to degass your mobile phase, then use high-purity helium gas only.

So I suggested that they replace their high pressure argon cylinder with a tank of high purity helium. Luckily they still had their original helium tank available so we hooked it back up. I sparged their mobile phase with the helium gas for about ten minutes then primed the pumps with the solution. The helium was left continuously flowing at a very low pressure (~ 2 psi) through a dedicated SS frit in the mobile phase. This keeps the level of helium in solution constant over time, resulting in stable baselines. After about five minutes the pump was running smooth and about as pulse free as you could hope for (0.1% pulsation). Lesson: Never assume anything and don't forget to make decisions which incorporate some basic scientific reasoning into them first.