Troubleshooting HPLC Gradient Valve / Proportioning Valve / MCGV GPV Leaks. How to Identify Them.

HPLC pumps which utilize low-pressure mixing VALVES are known by names such as: "Ternary" (3-solvents) or "Quaternary" (4-solvents) pumps.These types of HPLC pump configurations use a single, high-pressure pump head coupled to a multi-port / proportioning valve and represent some of the most popular and versatile pump configurations offered. Featuring random access to multiple solvent bottles (more than two is always better), lower operating costs and less maintenance work provides you with one of the best platforms to develop new methods on. I highly recommend them for most, but not all, HPLC applications (vs. Dual pump, high-pressure "Binary Pumps").

• If your HPLC system utilizes a single, high-pressure pump head coupled to a multi-port valve, then please remember that in addition to pump head maintenance, regular maintenance of the multi-port / proportioning valve is also required.

A few weeks ago I was hired by well known Pharma company to solve a gradient method problem that I was told has stumped their best scientists for almost one year. The client presented me with their validated UHPLC method which suddenly developed a shift in retention time of all peaks. The shift was significant, about 10% of the previous values over a 20 minute run, and had been observed on two different, but similarly configured HPLC systems in their lab. Changing the column to a new one showed no change on either HPLC system. They were out of ideas.

• Before I reveal the cause of the trouble, let us briefly think about what types of changes can result in a small, repeatable shifts of peak retention times. Four common ones that come to mind are: 

(1) Flow Rate changes;

(2) Column Temperature changes;

(3) Column Fouling;

(4) Mobile phase composition changes. 

Start the troubleshooting by ruling out the easy causes first (#1, 2 and 3 above).  

• (1) Flow Rate: When the actual flow rate is in question, start by measuring it manually Never trust the instrument's display screen value or the software's value for flow rate. Measure it. An easy way to measure the flow rate involves timing the amount of liquid that exits the HPLC detector line after a defined period of time. For example: If your flow rate is set at 1.000 ml/minute, then using water, measure the time it takes to fill a 10mL graduated cylinder to the 5 mL line. It must take exactly 5.00 minutes (= 1.00 mL/min). Run this flow test on each pump channel.
  
• (2) Temperature: The HPLC method should be run under controlled column temperature conditions. Verify this. Retention times are a function of temperature (i.e. cooler temps usually result in longer retention times, warmer = shorter). The temperature should be stable (~ 1 or 2 degrees C).
  
• (3) Column Fouling: To prevent fouling, wash the HPLC column with a solution that is STRONGER than the mobile phase after each analysis. Use fresh, clean solutions. Verify that the samples are dissolved in the mobile phase (100% dissolved) and filtered before injection. Verify that the injection volume is less than ~3% of the column volume and the concentration of the sample is not too high (avoid saturating or overloading the column). Solubility is very important for both the sample and any additives used in the mobile phase (to prevent precipitation). Anything that "fouls" the column support will directly effect the retention times and often the peak shape too. Be aware of these causes and take action to avoid them.  *Replacing a suspect column with a new one is often an inexpensive way of troubleshooting a "peak" problem. Always have a NEW spare column on hand for testing. *Columns are consumable items.
  

• (4) Mobile Phase: Changes to the actual amounts of additives, pH or final composition of the mobile phase may impact peak retention times (sometimes, the peak shape too). After all, the final composition used was developed for the purpose of establishing a reliable and reproducible method of analysis. It must be controlled. We must take steps to insure the mobile phase preparation and delivery are accurate. Always prepare fresh solutions each day (esp. all aqueous solutions!). pH values may change after a few days (e.g. even in MeOH / acidic solutions), bacteria/mold/algae grow quickly in many solutions, even in the refrigerator, so only prepare what you need for the day. Evaporation of more volatile solvents (in pre-mixed solutions) can change their actual concentration (always protect them from heat and evaporation).
  

*There is another way that the mobile phase composition can change which often goes unseen. It can change during delivery to the column. The HPLC's low pressure proportioning valve that allows us to easily select and use different solvents can develop small internal leaks, resulting in valve cross-flow leakage. This cross-flow leakage allows liquid (or air, if the line is not connected) to be drawn out of one channel and into another, changing the actual mobile phase composition. This happens because the valve seals, esp if they have been left unused for a long time, can change shape (e.g. shrink) and begin to leak over time. Often the amount of leakage is very small (ul/min), but depending on the method, a small change may result in a significant change to the chromatography.

I reviewed the client's method parameters and concluded that the method met good chromatography fundamentals. Checking the flow rate (using a graduated cylinder) confirmed the flow rate was accurately shown. A review of their mobile phase preparation procedures and methods also appeared OK. Degassing of mobile phase and column temperature were also satisfactory. 

As I looked more closely at the two running HPLC instruments they used, I began to quickly zero in on the most likely problem. 

• A long stream of air bubbles were observed exiting the HPLC pump's gradient valve leading into the high pressure pump head, but no air bubbles were seen exiting the degasser's outlet line (IOW: The vacuum degasser may or may not be the cause, though it is critical to insure the degasser is clean and fully serviced before use. Have the degasser professionally serviced first before proceeding with troubleshooting. Using a damaged degasser will make it difficult to use the pump or run any valid tests as degassed solution is needed). This was observed on several of their HPLC systems, including the two used for this method. The fittings connecting the lines from the degasser module to the valve were correctly connected (as a loose connection would cause air to leak in and must be quickly ruled out). 

The cause was from one or more of the unused gradient valve positions leaking air into the flow path, changing the mobile phase composition. Of four possible mobile phase lines available (A,B,C,D), the client only had two lines connected to mobile phase bottles (A,B) with the remaining two lines left open to the air. The internal valve seals in the unused 'C' and 'D' valve positions had deformed, shrinking in size, sticking,leaking, allowing air to flow into the mobile phase on one of the channels. This resulted in a change of the organic composition % used in the method (due to a cross-flow leak), changing the peak retention times (as the actual mobile phase composition used in their gradient was different). I directed the HPLC pump's outlet line to waste, placed all of the solvent pickup bottle lines (A,B,C,D) in a beaker filled with IPA and allowed the pump to run pure IPA at 1 mL/min across each channel, one-at-a-time (100%), for ~ 20 minutes to re-hydrate the internal gradient valve seals. This was repeated with each valve position, then all of the lines were placed in fresh mobile phase solution, primed and flushed. The system was restarted and the method now ran showing the expected peak retention times. Instructions were provided which included regularly using all of the channels and valve positions plus flushing weekly to maintain valve operation. Use ALL of the lines and flush the valve(s) through all positions, one-at-a-time, on a regular basis. If prolonged flushing with pure IPA does not fix the leak, then it is time to replace the valve. All valves eventually wear out and must be included in maintenance inspections and checks. This is especially true when you purchase your HPLC system at an auction or from an 'equipment' reseller. Never assume that the 10+ year old HPLC valve is OK. Test it first (e.g. Acetone tracer test).

 

Acetone Tracer Test: If you suspect that a cross-flow leak exists on a gradient valve, then one method I use to check for leakage is to mix up a "Tracer" solution of pure organic (often ACN) that has 1% Acetone mixed in (for RP methods). Remove the column and replace with a restriction capillary. Place the tracer solution on the valve position you suspect may be leaking at an appropriate flow rate and set it for 0%. Run one of the other channels with 100% (pure ACN in this example) and monitor the UV (265nm) for the presence of acetone. If the acetone leaks into the channel you are using, it will be easy to observe on the UV trace. So called "bubble" tests (introducing and monitoring the position of a gas bubble into the low pressure solvent line) are not reliable leak detection methods for small leaks. Use a tracer such as acetone to find the leaking channel(s). You can read more about these types of Valve Leakage tests in this article (Click Here).

Tips and Advice for Priming your HPLC PUMP (or similar pumps, FPLC or UHPLC Pump)

The single most important component of any HPLC system is the Pump module. We often refer to it as "the heart of the HPLC system". 

• You may have the most sensitive HPLC detector, the best column, a perfect method of analysis, but none of this will matter unless the HPLC pump(s) that provide mobile phase to the system operate perfectly, all of the time. If you have a poor quality (or poorly maintained) system, then you will spend much of your time trying to establish reliable flow through the HPLC system, not running samples. 
• Before using an HPLC system, you should prime all of the lines in your HPLC pump. This is needed to purge any air from the tubing, introduce fresh mobile phase to each line and then to VERIFY that each channel delivers the reported amount of fluid to the column (measure it).
  

• NOTE: This is a LONG, detailed article with lots of information, Hints and Tips. It is available in PDF format for download, here.

The HPLC pump's ability (stability) to provide reliable operation depends on: 

(1) The Chemical, Physical and Miscibility properties of the Liquid(s) being pumped;

(2) The Amount of dissolved gas inside the liquid (must be minimized);

(3) The Temperature of the room (or HPLC) must be stable;

(4) The Position of the mobile phase bottles (relative to the pump, above or below);

(5) The Solvent Pickup Filters used (are clean and appropriate in material & porosity);

(6) The Fittings used are correctly installed & tightened;

(7) The types of Tubing used are chemically, temperature and pressure compatible (esp. the Inside Diameter of the tubing);

(8) The Selected Flow Rate(s) and Back-pressure are within the optimal range of the pump;

(9) All mobile phase solutions are Filtered, Freshly prepared and Degassed;

(10) How often the Pump is properly Inspected, Cleaned & Serviced.

 The HPLC pump is the most important part of your HPLC system. Take care of it. Neglect or abuse it, and you may lose time and money. Almost every problem you experience using an HPLC will be related in some way to the pump. Make sure you understand the flow path of the system in detail, and have the training to setup and use it properly. Take a hands-on training class (not a video or web based tutorial) to learn how to use the pump on your specific HPLC system. Learn how to run simple verification tests to check the flow rate (best done with a graduated cylinder). Never rely on the software values, check and verify everything yourself. Priming and flushing are needed any time air bubbles are present, mobile phase solutions are changed or the system has sat unused (this includes overnight). Always flush multi-channel pumps and valves (i.e. Binary, Ternary, Quaternary...) using a setting of 100% channel composition. Run one channel at a time at 100%, not 25% or 50% to flush channels (a common novice mistake). Flush ALL channels on a regular basis.

OK, so what can you do to make sure your HPLC pump is properly primed with fluid and operating to the best of its ability?

Start, by reading the operator's manual for your pump. Review the procedures for connecting it to the system, become familiar with the flow path and understand the procedures to prime the pump heads. Practice these procedures.

If an inline vacuum degasser is used, become familiar with the specifications, chemical compatibility (some are not compatible with solvents such as strong acids, strong bases, THF, chloroform, fluorinated additives and so on) and internal channel volume of each chamber used. It is useful to know what the degasser chamber volume is to figure out what the total channel priming volume is. This may be different for similar systems. Check, measure, verify, do not assume.

Priming Volume: The total volume contained in each channel's low-pressure line from the mobile phase bottle to the degasser + the degasser chamber channel volume + the total volume in the line from the degasser to the pump head (or multichannel valve) = the total minimum volume you must flush out before using the system. Because flushing just the minimum of volume (1x) of fluid through the channel is unreliable, flush 2x, 3x or more times this total volume, per channel (or as much fluid as it takes), to prime each channel. *If no degasser is present, then just calculate the volume contained in the low pressure tubing from the bottle to the pump head/valve. Set the pump to direct the flow to waste and use a high initial flow rate to speed up the priming process.

Use fresh mobile phase (prepared daily and filtered). Make sure the solvent pickup filters are clean. If possible, have the bottles placed higher than the pump's inlet (once flow has been established, this will allow natural siphoning to push liquid towards the pump head). Prime all of the lines used. The pumps run on liquid, not air so try and fill any of the lines with pure mobile phase before you connect them to the pump and/or degasser (If all of the lines are prefilled with fresh liquid, you can skip this part).  

There are two ways to PRIME EACH line (Flushing the Channels).

• *First, open any Prime/Purge or Waste Valve so the mobile phase is directed to waste, not the injector, column or detector. Our goal is to initially fill the lines with liquid, quickly, and we do not want these fluids to go through the entire HPLC system (i.e. column), just the HPLC pump.

(1) Wet Priming use a syringe fitted with a Luer-to-threaded fitting adapter (usually 1/4-28) to draw liquid through the tubing in the mobile phase bottle and into the pump's degasser and/or pump head's inlet. Be sure to have this type of syringe available (very useful). Never push fluid, only draw fluid through the tubing, just like the pump does. Connect the syringe to the mobile phase bottle lines, degasser ports and/or pump head multichannel valve or pump head inlet, as needed, to draw liquid through until all lines are filled.

(2) Dry Priming using the HPLC pump to draw the mobile phase out of the bottles, through the lines, degasser channels and to the pump head or multichannel valve. Note: "Dry" because the lines (low pressure tubing) are initially dry when we start. Always do this one channel at a time (e.g. A = 100%). This insures no miscibility or mixing problems and is standard procedure. Start with a modest flow rate to get the fluid moving through the lines, then increase the flow rate to speed up the process. The low pressure Teflon tubing is transparent so you can watch this process. Repeat with each channel. Note: Some HPLC pumps will struggle to perform this type of dry priming, as they will be unable to draw the liquid up from the bottle and/or pump the air out of the system. Pre-priming the lines using a syringe (as in #1 above) will help solve this. Running the pump with just air inside the lines may result in increased wear on the system (esp the piston seals) so if the system struggles to fill with liquid after one minute, discontinue and manually wet prime each line.

NOTES: 

• The back-pressure shown on the system readout should be very low during this initial  priming process (e.g. < 15 bars) as the HPLC system should not be plumbed with the column or detector inline, during the priming process (it should be by-passing those parts). Only the viscosity of the solution, the selected flow rate and the internal diameter of the tubing going into and out of the pump will contribute to the observed back-pressure, and this should be very low value.

• Once you have verified that liquid is exiting through the pump head waste port, you can increase the flow rate to speed up the priming process, but pay attention to the back-pressure. It should increase as the flow rate increases and drop as the flow rate drops. Continue to prime each channel in this way, one-at-a-time, until all channels are primed and flushed with liquid. You can gradually slow the flow rate down as you stop, to transition from one channel to another.
  

• If liquid has been drawn to the pump head, but the pump head still is not pumping liquid through it, it may be experiencing cavitation (air locked). If there is an outlet port on top of the pump head, the outlet fitting above the pump head can sometimes be briefly loosened with a wrench, allowing the system to push the air out (open it slightly with a wrench, then quickly close it after liquid comes out). Have a towel ready to soak up any fluid that comes out. Keep the area clean and dry. Alternatively, try drawing liquid through this port, while it is running, to gently fill the pump head chamber and remove the air.

• In some case, the inlet or especially the outlet check valve(s) can also become "stuck" open. When buffers are left in the system (they should be flushed out with water), crystals and particulate matter may deposit on the valve resulting in poor sealing, leaks or air being drawn through. Drawing liquid out of the pump head's outlet port with a syringe (or gently pushing it through the pump head) may remove the air bubble, debris and prime the valve, restoring function. Note: If needed, shut down the pump and clean/replace any contaminated or worn check valves before proceeding.
  

• In more extreme cases, you can change the mobile phase going into the pump head to a more viscous intermediate solvent to get things moving (an alcohol such as IPA might work well. If buffers have been used, then always first flush with pure water). 

• Degas all eluents / mobile phase solutions used. All of them. Degassing will help reduce the formation of bubbles inside the pump head. Failure to properly degas the solutions used may result in loss of prime, baseline and/or pressure instability. Make sure your degasser is operating properly (electronic vacuum degassers only last ~ 5 years at most. Be sure to have them professionally serviced). Sonicating fluids at the bench or using vacuum filtration to initially remove gas from the solution will only degas the solution for a short time (minutes). Gas will slowly diffuse back into the solution resulting in baseline noise, drift and pump problems (for HPLC, only use inline degassing or Helium sparging).
  

• Verify the flow rate. It may be unwise to rely on the indicated flow rate shown on the instrument screen or display. It is wise to measure the flow rate of each channel, separately, using a graduated cylinder and a timer. This is the most reliable way to determine what the actual flow rate is through the system (and is also the method we use during performance verification or qualification testing too). To check the flow, make sure the system has been primed and flushed. Install a flow restriction capillary in place of the column (to provide the required back pressure). Set the flow rate to a value which is appropriate for the pump and measure/record the volume delivered vs. time. Example: Using a flow rate of 1.000 mL/min obtain a 10 mL volume, glass laboratory grade graduated cylinder. At time zero, direct the flow from the restrictor's outlet into the graduated cylinder. Measure the volume of fluid collected in 8 minutes. *It should be 8.00 mLs.
  

If you continue to have priming problems and/or air bubbles disrupting the flow there are four more things you can check. 

1. Make sure the solvent pickup filters/frits are clean and unobstructed (these are maintenance items). If the filters are obstructed, then a vacuum may form on the line resulting in pump cavitation and loss of prime. One quick way to check if this might be a problem is to remove the suspect solvent pickup filter from the tubing, then try again. If flow is restored w/o the filter in place, then the filter may have been clogged. Install a new solvent filter as soon as possible. *Never run the HPLC without solvent filters installed. Those filters perform a very important job and protect the flow path of the system.  
2. Service the Pump Heads. Regular cleaning, inspection and replacement of worn parts must be done to maintain the function of the pump. Worn parts will result in failures, instability, lost time, plus invalid data. The pump has many mechanical parts which wear out and require replacement. Most pumps should be inspected/serviced every 6 months. Keep the pumps clean and fully serviced (replace: piston seals, pistons, frits, check valves as needed). Depending on the brand, model and applications, the types of parts needed and the frequency of repairs varies widely. *This is discussed in another article. 
3. If your HPLC system has an inline vacuum degasser (either a standalone or integrated module), it may be damaged, contaminated or broken. The typical service life of an electronic inline vacuum degasser is only five years (some models have even shorter lifespans). Degasser's with internal damage may result in contamination of the mobile phase. A failing or damaged HPLC vacuum degasser may directly contribute to analysis problems (ghost peaks, pressure instability, poor baseline stability...). Have your degasser professionally diagnostically tested and serviced often.  
4. Clean and/or replace any worn or damaged inlet or outlet pump head valves. Not flushing buffers out of the HPLC system on a regular basis or remain in contact with the solution for long periods of time can damage the valves. In some cases, cleaning is all that is needed, but in others, replacement is required to restore function. Be sure to have the system professionally serviced on a regular basis.
   

• Additional Troubleshooting Info can be found here:

Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)

HPLC to UHPLC Conversion Notes (Gradient Time Program Adjustment)

In an earlier article we discussed how to adjust the flow rate, injection volume and column dimensions when scaling an HPLC method UP or Down. The formula's needed to do this are fairly simple. If we adjust for changes in the column dimensions or flow rate, what types of changes are needed to adjust the gradient time? The formula to make this adjustment is also very simple. Here is the information you need.

Terms Used in Formula:

Time in minutes, Gradient (Initial): Tg1

Time in minutes, Gradient (New):   Tg2

Flow Rate in mL/min, Column (Initial): Fc1

Flow Rate in mL/min, Column (New): Fc2

Column Diameter, mm (Initial): Dc1

Column Diameter, mm (New): Dc2

Column Length, mm (Initial): Lc1

Column Length, mm (New): Lc2

• Tg2 = Tg1 x (Fc1/Fc2) x ((Dc2²) / (Dc1²)) x (Lc2 x Lc1)

Here is an example problem to solve for. 

If we start with a flow rate of 1.000 mL/min (Fc1) on a 4.6 x 250 mm column with 5 micron support (Dc1 & Lc1) and have an initial Gradient Time of 10 minutes (Tg1), then what would the new gradient time be if we switched to a sub 2 micron support in a 2.1 x 50 mm column (Dc2 & Lc2) at 0.200 mL/min (Fc2)? 

To solve the equation we will plug-in the values for each part of the equation separately, then multiply them to obtain the result.

  (Fc1/Fc2):    1.000/0.200 = 5

  (Dc2²) / (Dc1²)  4.41/21.16 = 0.21

   (Lc2 x Lc1) = 50/250 = 0.20

  Tg2 = 10 x 5 x 0.21 x 0.20 

  Tg2 = 2.10 (or 2.10 minutes)

If a 2.1 x 50 mm column was substituted for the 4.6 x 250 mm AND the flow rate was changed from 1.000 mL/min to 0.200 mL/min, then the initial programmed gradient time of 10 minutes would be changed to 2.1 minutes

UHPLC TIP: Reducing the Column Temperature to Offset Frictional Heating Effects (Causing Poor Resolution)

HPLC column temperature is a critical variable that we adjust and optimize during method development. We use it as a variable during the method development process to improve solubility, optimize peak shape and increase resolution. Once established, it must be carefully controlled during the method analysis to provide reliable and reproducible analysis results. Change the column temperature and you may also change the results obtained. This is a fundamental method development tool and must not be forgotten.

If you are developing a new UHPLC method OR perhaps scaling an HPLC method to utilize 2.5 micron or smaller support particles, then you may observe a loss of resolution or poor peak shape in the new method. There are many reasons why this may occur, and the most common ones relate to not optimizing all of the method parameters correctly when scaling the method (e.g. dwell volume too large, flow cell volume too large, injection volume too large, sample rate too slow, flow rate not optimized, mobile phase composition changes not in scale with the gradient...). But there is another reason...

Resolution may be reduced or lost when all of the initial scaling and instrument set-up parameters are optimized. What is the most likely reason for this? In many cases the use of substantially higher flow rates (relative to linear flow rates) and the use of smaller diameter particles results in much higher backpressures (you may recall that if you halve the particle size, the backpressure increases 4x). The resulting backpressure might be 2, 3 or even 4 times higher than observed in the original method. While these higher backpressures were well within the operating parameters of the HPLC system used, the results obtained were poor. The possible cause? The much higher backpressure increased the amount of frictional heating inside the column, raising the actual analysis method temperature and changing the separation conditions. 

Pushing mobile phase (liquid) through a chromatography column generates heat and pressure. The heat generated increases the actual temperature of the column and reduces the viscosity of the fluid. In conventional columns (i.e. 4.6 x 150 mm, 5u) at 1.00 ml/min, this heating effect is minimal, but at much greater column pressures, > 400 bars, the frictional effects may be substantial. These types of very high pressures may be seen with methods which utilize columns containing the smallest particles (1.9 to 2.5 micron). Enough to change the temperature in the column by several degrees (e.g. >5 degrees C) and result in different method conditions. So, what can you do about this? The most direct way to address the problem is to run the same method at a lower temperature (perhaps decrease by 5 C to start with). This will slightly raise the backpressure (lower temperature equals higher viscosity), but it should cool the column and restore the original temperature conditions used. Additionally, we suggest that you always start column equilibration using a flow ramp to gradually increase the flow over time and reduce the overall heating effect and resulting "shock" placed on the column. An initial delay at equilibration may help reduce these effects (gradually ramp up to the regular flow rate and hold). You may need to try several temperatures and this may be easiest to do if your HPLC has a column compartment with heating and COOLING capabilities. Optimizing the temperature and internal pressures may increase the column lifetime and result in better overall data reproducibility.

Modern HPLC Method Development Tips (PART II):

This is the second of two articles (Part I) which will provide suggestions on how to improve the HPLC (UHPLC) method development process. - PART II

INITIAL METHOD DEVELOPMENT:

1. Before you start, learn what you can about your sample, its hazards, solubility and properties. Conduct a quick literature and/or keyword search on the web using a popular search engine (e.g. GOOGLE). You can often find many journal articles, white papers, application notes and chemical data on the web in just a few minutes.
2. Determine which liquids your compound(s) are soluble in. Use a pipette and several glass vials or tubes with different solutions (pH is important too). This will narrow down the types of mobile phase and chromatography modes that you can use.
3. Column choice is the most important part of method development. The stationary phase that you choose has the single greatest effect on selectivity! Select the right column and mode of chromatography. Most RP methods should start with at least a modern ultra high purity, metal free type B silica column with a C8 or C18 support (*But you must select the column type that best suits your application). For small molecules (<1,000 Daltons) select a standard sized analytical column with a 2.5 to 5.0 micron particle size and pore size between 60 and 120 Å (e.g. 4.6 x 150 mm; 3.0 x 100 mm). *For larger peptide or protein molecules you will need a particle with a large pore size (~ 300 Å). For optimal results, columns with very small internal volumes should be paired with HPLC systems with similar ultra-low internal delay volumes. Your HPLC system should be optimized to balance the needs of good mixing, low delay volume and proper sampling rate for your method. If the method is likely to be transferred to different types of HPLC systems, then you may want to initially stay away from the < 2.1 u particle supports for your method development. At this time, these tiny particles can not be reliably packed into columns as well as the larger sized particles and generally have much poorer %RSD values than larger particles (i.e. 2.5 to 5 u). Method development is initially easier & generally more rugged using conventional particle sizes [Keep things simple when starting out. You can always change later on]. Once you have selected a column and decided on a flow rate, make sure you calculate and measure the actual column void volume to find the 'Tzero value' (column dwell volume). You will need this value to find out if your compound has been retained on the column (K prime) and to also determine most of the needed performance calculations and parameters.
4. Once you have selected an HPLC column (if possible, please start with a brand new column) you will need to test it and establish a baseline to show that it meets both the manufacturer’s specifications and, far more importantly, that it meets your method’s requirements. In most cases, do not use the manufacturer’s QC test solution for this. Those stds are designed to allow the column to easily pass manufacturing QC, not the more critical requirements that you may need to prove. We prefer to use a real sample mixture (~2 compounds) that are similar to the type proposed for the method. They must be well retained on the column (K prime of > 2), easy to prepare, stable and reliable. This std and the specific method you develop for it, will be used to prove the column’s performance (i.e. R, S, K prime, Tzero, Plates). It will be used again, when the column’s performance is called into question.  
5. Notes on Mobile phase and additives: Keep it simple. Avoid the use of any additives such as ion-pairing reagents when first starting out. They are overused in general and can cause problems later on. If required, they can always be added later on. Use only HPLC grade solvents, fresh RO HPLC grade Water and the highest purity acids, bases and/or additives, if required. Use only filtered (0.22u) products. Always dissolve samples in the mobile phase (or a weaker solution) for injection. For many RP methods a low pH mobile phase (~ pH 2.5) provides a good starting place. Samples with a pKa far enough away from this value are likely to be retained, stable and unaffected by small changes in pH. *pH is usually one of the last parameters which is optimized.
6. Use a Gradient Method to find the approximate elution conditions of the mixture AND make sure you are detecting all of the compounds injected onto the column [For dedicated RP isocratic methods, start with a high organic mobile phase % (i.e. 95%) and record the results. Continue to reduce the organic content in steps of 10% and observe where the best compromise exists between retention and elution]. For RP gradient methods, start with a very high aqueous % (e.g. 98%) and run your gradient, slowly, to a very high organic % (e.g. 95% or 98%, not 100%!) to make sure you retain, hold, and then elute everything. Your mobile phase conditions must be strong enough to elute everything off the column during the gradient portion of the run (long enough hold time). Please Do NOT include the gradient reversal portion back to initial conditions (wash and re-equilibration) of the gradient as part of the same analysis method. The method should end after the gradient has reached and been held at its maximum level for a period of time (we refer to this as the "hold time"). It should NOT switch back to the initial conditions to re-equilibrate the column. Don't make this novice mistake. Column flushing and re-equilibration should be a separate method and/or step, separate from your analysis method. If you include your column flushing (washing) and re-equilibration steps as part of your analysis method, you are also forcing the baseline's slope to change radically. This may interfere with integration plus include extra peaks and baseline changes that you are going to have to integrate, identify and explain to others (e.g. auditors). Additionally, including these wash steps as part of the same method wastes time as you must wait for these steps to complete before you can start to process the data obtained in the analysis method. Summary: Develop methods like the professionals do and create separate Analysis and Flush/Re-Equilibration Methods (or steps).
7. If your compounds can be seen with a UV/VIS detector, then make sure you are using a Diode Array Detector (aka, DAD or PDA) set to scan a wide range of wavelengths (e.g. 210 - 410 nm). Method development should not be performed using a single or multi-wavelength detector. This invites errors, limits the utility of the method and does not result in any cost savings. You must have a full scanning detector so you can detect all possible peaks at the same time. If you use a single or dual wavelength detector you may not know if an impurity has been introduced or if you have a co-eluter (because you will have no way to detect it). Generic starting settings for Wavelength Bandwidth should be ~ 8 nm, software based Reference Wavelength OFF and the sampling rate must be set to collect at least 20+ peaks/sample peak of the narrowest sample peak observed and integrated (at ½ height). Run with scanning turned 'on' for all analysis methods and review the spectral data for each run. This provides important qualitative data about the compounds which may be used for purity determination and also to demonstrate how the method is selective for the compound(s) of interest. *If your compounds do not absorb well (weak chromophores), then you may still want to have a UV/VIS detector inline (first) with a secondary detector second. The UV/VIS detector will still be very useful for troubleshooting and detecting other compounds. Select an appropriate type of detector and compatible mobile phase as required for the secondary detector (e.g. RID, EC, FLD, ELSD, CAD, MS...). Note: If available, LC-DAD-MS is one of the most useful instrument setups for LC method development.
8. This needs to be repeated (from Part I)....  Before starting ANY HPLC analysis, the HPLC pump must be running and operating with no problems, achieving a stable baseline, steady flow rate with as little pulsation as possible (~ 1% ripple or less). Accuracy depends on this. Do not begin any HPLC analysis unless the HPLC pumping system is working perfectly.