Troubleshooting a GRADUAL HPLC PUMP PRESSURE INCREASE OVER TIME (When PURGING, DURING an ANALYSIS or when FLOW is DIRECTED TO WASTE)

A fully equilibrated column at a constant temperature and flow rate should result in a stable back-pressure over time (~1% variation). Have you observed slowly increasing HPLC system back-pressure readings, even when you are fully equilibrated and everything should be stable? Read on to find out why this may be happening...

First, you must know what are "normal" or expected values for:

• HPLC Pressure (and normal changes over time);
• Baseline changes (e.g. drift, equilibration or normal rise/fall);
• Peaks (e.g. Sample peaks vs. Valve position peaks, spikes or Noise);
• Retention time delays (due to a leak, gradient delay, fouled column etc).
  

  vs. those that result from an actual hardware faults. To operate any HPLC system, you must learn how to identify these. It will take many years of hands-on practical experience.

If you know what to look for, the HPLC system will provide you with clues when something is wrong. "Pressure" is one of those clues... Learn to always pay special attention to the system back-pressure and watch for signs of trouble. Pressure should change in a predictable way.

• HPLC system pressure is not a variable in HPLC method development, but it is an effect resulting from forcing liquid through a highly restricted flow path. 

Always monitor the HPLC system pressure under all conditions (e.g. analysis, washing columns, equilibration, flushing to waste). When the pressure changes, verify it changes in a predictable manner. Acceptable real-time System pressure depends on: (1) the flow rate; (2) the mobile phase composition; (3) the temperature; (4) the flow path selected (e.g. valve switching, running through the column or to waste). As the column becomes fouled over time, the pressure observed may also change (increase). 

• *COLUMN fouling is one of the most common reasons for the system back pressure to slowly rise over time (usually over weeks) for the same analysis method. Review the sample preparation, injection solvent choice, miscibility/precipitation and/or concentration levels to find the problem.
  

If you change the tubing connections or actuate a valve, (you change the flow-path in doing so), then the pressure observed may also change too. 

Let us consider what other areas of the HPLC system may change the system pressure.

Pump Filters: Most HPLC pumps have a small disposable outlet filter installed at or near the pump outlet line (Note: In the case of most Agilent brand HPLC pumps, a small PTFE filter may be found at the pump's outlet valve or inside of the prime-purge valve). This filter is designed to collect any piston seal debris or other large particulate contamination from entering the rest of the HPLC system's flow path (i.e. the injector, column, detector...). These small filters (~ 10 to 20um) collect and retain the debris inside the filter so it does contaminate or obstruct the flow path down stream. It is not designed to filter your mobile phase for you (You should have pre-filtered all solutions used in your HPLC). However, this accumulated debris slowly results in a partial obstruction of the flow path, increasing the overall system back-pressure. This may not be obvious to a new user running an analysis method, but the pressure increase due to the clogging filter will occur slowly over time, often masking the change. In a month, it may represent 10-20+ bars increase. In a clean system, if you redirect the flow from the column to waste, you should observe the system back-pressure drop to just a few bars (maybe close to or near zero, depending on the viscosity of the solvent and flow rate). You should know what the "normal" pressure is when the system is directed to waste for many commonly used solvents at typical flow rates. Knowing these values will help you troubleshoot many problems in the future.

• Example: With a new pump outlet filter installed in most 'standard' HPLC pumps, pure ACN solvent directed to waste, running at 1.00 mL/min may show a reading of about 7-bars. If one week or one month later the reading changes to 15-bars, then the filter is clogged with debris and should be replaced. *Perform this type of check on your HPLC pump every day. What is the "normal" back-pressure reading when you direct your typical mobile phase to waste ? What is the value for pure Methanol, ACN, Water, IPA etc. ? It will be different for each HPLC system.
  
• Do you use Aqueous Mobile phase? If so, please filter the final solution through a 0.45 micron (or 0.22u) filter before use. We have observed many laboratories using non-HPLC grade water (e.g. Distilled Water or Sterile Water) resulting in plugging of these pump outlet filters. Always use fresh HPLC grade water (i.e. RO Water) for RP analysis and when preparing buffers.
  

While equilibrating a mobile phase for an analysis the system pressure should stabilize at some point, and also return to the same pressure range after the analysis is complete and the system is allowed to fully equilibrate. As a matter of fact, you should be monitoring the system pressure and detector output after each analysis and wash to determine when the system is ready for the next injection. If the system does not stabilize over a reasonable amount of time, but instead shows a gradual increase in pressure (over the course of minutes, hours or one day), then this may be a sign that their is a partial obstruction inside the HPLC system. While there are many places a partial obstruction could occur (e.g. the injector or column), one of the most common and easy to check for areas is within the pump's outlet filter. Check by diverting the flow to waste and record the system back-pressure. If it is higher than what is expected, the outlet filter should be replaced first. Note: Other problems such as clogged mobile phase solvent pickup-filters or even worn piston seals may also show similar pressure increases too, but most of the time the pump's outlet filter is the cause.

Conclusion: 

• REPLACE the disposable outlet filter found in the HPLC PUMP EVERY MONTH. 

Yes, every single month. These are inexpensive disposable filters designed to protect the flow path of your HPLC system. This is one of the least expensive consumable parts that can have the greatest impact on overall HPLC performance. Stock plenty of these filters and learn how to replace them. Your baselines will be more stable allowing for better quantitation, higher sample through-put, less down-time and less service.

1. For many of the the Agilent 1050, 1100, 1200 and some 1260-series modules using the classic style pump heads, P/N  01018-22707 is suggested ($8.50 USD each). *Please refer to your pump manual to find the correct number for your brand and model of HPLC pump.
   

Agilent Quaternary Pump (e.g. G1311A ) "Secret" Operator Tip to FLUSH the HPLC Pump in 1/2 the time!

One of the most popular "tips" taught in our Agilent 1100 and 1200-series HPLC training classes shows users how to speed up the daily priming and flushing process of the Quaternary Pump. Many people use these pumps without taking advantage of the Quaternary pump's higher flow capability. If you are not currently using the higher 10mL/min flow rate capability offered by this pump (vs. the Binary pump's 5 mL/min), then you are missing out on a free time saving feature. Please read on to learn how to use this feature.

Based on the HP 1050 pump and introduced in 1995 as the "1100-series" version, the G1311  "Quat" pumps are one of the most popular research grade HPLC pumps found in laboratories today. They are extremely reliable, rugged, easy to operate and service. The Quat pump is driven by an easily accessible, single pump head with an in-series, servo controlled dual plunger and Multi-channel Gradient Valve ('MCGV') for 4-channel solvent proportioning with an active inlet valve (known as the 'AIV', first used in the HP 1050 pump and the reason for this pump's high reliability. No more "sticking" inlet valve issues!). Unlike the Agilent Binary pump (G1312), which uses two separate dual plunger pumps (2-channel) at up to 5.0 mL/min (maximum), the Quat pump offers an extended flow range, up to 10.0 mL/min (maximum). However, most users are not aware of this or do not know how to utilize this higher flow rate feature because the Quat pump defaults to a maximum flow rate of 5 mL/min at initialization. The ability to program the pump to operate at flow rates greater than 5 mL/min requires a "trick" to activate it (which apparently is a secret as we rarely encounter customers who are aware of how to use it). 

Let me share with you why you would want to use this feature, why the feature is hidden to most and of course HOW TO ACTIVATE IT on the Quat pump.

• Q: Why would you want to run the pump at 5 to 10 mL/min? Semi-prep columns can be run within this flow rate range, but a more common reason to operate at 10 mL/min is for daily system start-up. Anytime you replace or change the mobile phase bottle/solution OR when you startup the HPLC system (each day) one of the very first things you need to do is prime or flush each of the mobile phase channels, one-at-a-time through the system to waste. Air bleeds into the system when it is not used and this procedure primes the lines and pump head with fresh mobile phase preparing it for use. The system's flow path is directed to waste (via the open, prime-purge valve) during this step so back-pressure is not a concern. The higher the flow rate you can use for this flushing step, the sooner you can complete it. If you run the pump at 10 mL/min vs 5 mL/min, then flushing can be completed in half the time. This is especially useful if you have a model G1322A degasser module installed as the internal volume of each degassing channel in the G1322A is 10-12 mLs, requiring extended flushing times (4x channels = 30+ mLs flush per channel) before moving on to the next channel.

• Q: Why does the Quat pump initialize with a reduced, 5 mL/min maximum flow rate? The Quat pump was designed to meet two different operating pressure ranges. From 0 to 5 mL/min the permitted operating pressure range is 0 - 40 MPa (0 - 400 bar). Above 5 mL/min, the operating pressure range is reduced, 0 - 20 MPa (0 - 200 bar). As most analytical chromatography is performed at flow rates below 5 mL/min, the system initializes using the more practical, 0 - 400 bar range, limiting flow rates to 5 mL/min maximum. The default maximum pressure field is set to 400 bars. You should always change the maximum pressure value from 400 bars to a more realistic maximum pressure (lower value) for your method. Use a maximum value that is appropriate for your own method. *The only time you will want to set it to the maximum value is when conducting a Pump Pressure/Leak test (it must be set to max pressure for testing).
  

• Q: When I try and enter a pump flow rate larger than 5 mL/min, the system does not accept it. How do I program the pump to increase the flow rate past 5 mL to 10 mL/min? In order for the system to accept a flow rate of greater than 5 mL/min, you must FIRST set the maximum pressure limit to a value that is 200 bars or less (within the allowed "0 - 20 MPa (0 - 200 bar)" range). Once the maximum pressure limit has been reduced in the method, the system will then allow you to enter a higher flow rate such as 9.999 mL/min (10 mL/min). As long as the maximum pressure alarm is set within this window (200 or less), the pump will allow flow rates above 5 mL/min to be used. Now you can program the pump to flush lines or prime the system at twice the speed of the Binary pump equipped systems (10 mL/min).

Please share this "trick" with other users of the G1311A, G1311B, G1311C versions of this pump so they can maximize their time and productivity. Let us know if you find this tip useful.

Backpressure Changes, Pressure Drop from HPLC Tubing Selection (0.007, 0.005, 0.010")

In previous articles we have discussed how the choice of column particle size directly changes the system backpressure. Smaller particles generate higher back-pressures. We have also discussed the importance of HPLC tubing selection to minimize delay volume and diffusion within the HPLC's laminar flow path. Let us now focus on how the tubing's internal diameter and length impacts the total HPLC back-pressure (or pressure drop) observed. 

Key Points:  

1. Try to optimize the plumbing of your HPLC system.  
2. HPLC Tubing lengths between connections (or HPLC modules) should always be as short as possible. 
3. Pressure drop is dependent on the tubing length and inner diameter. Doubling the inner diameter of the tubing will decrease the pressure by a factor of 16.

Once the HPLC tubing connection lengths have been minimized, the next critical dimension which affects band broadening, delay volume and peak-width is the internal diameter (ID) of the tubing. The tubing selected should be narrow enough to reduce the undesirable spread of the peak(s) inside the tubing, but not be so narrow or restricted to result in clogs or obstructions (which is why good chromatography guidelines should be followed insuring that each sample is fully dissolved and filtered before injection). Commonly used tubing ID’s for most analytical HPLC systems are: 0.010” (0.25 mm), 0.007” (0.17 mm) or 0.005” (0.12 mm). By far, 0.007” (0.17 mm) is the most commonly used size for modern analytical HPLC analysis as it offers a compromise between low delay-volume and modest back-pressure (with fewer clogs). However, in addition to the much lower internal volumes which accompany the narrower ID’s, the pressure drop measured across equivalent lengths of tubing may change dramatically and this should be noted during set-up, selection and operation. Take the time to learn what "normal" backpressures are under specified conditions.

 

Understanding how the HPLC system backpressure changes as the internal diameter of the tubing varies is extremely useful in troubleshooting a number of common HPLC problems.

Let us compare the pressure drops measured across three popular HPLC tubing ID’s of the same length (40 cm) using common HPLC mobile phase solvents. This table will help illustrate the observed backpressure changes that the tubing ID and liquid have on the pressure drop.

PRESSURE DROP (in bars):

SS Capillary Tubing, 40 cm length, flow rate 1.000 mL/min.

Mobile Phase / Tubing ID	Water	ACN	MeOH	MeOH/Water (1:1)	IPA
0.010” (0.25 mm)	0.7	0.2	0.4	1.2	1.5
0.007” (0.17 mm)	2.7	1.0	1.6	5.1	6.2
0.005” (0.12 mm)	10.4	4.0	6.3	19.1	24

Note: Pressure drop is also a function of tubing length so if we halve (1/2) the length of tubing used, we also will reduce the pressure drop by one-half. 

Note the four-fold change that narrowing the tubing ID has at each ID reduction. The change is more dramatic when viscous solutions are used (i.e. MeOH/Water or IPA). If you re-plumb any part of your HPLC system with new tubing, then awareness of this physical change will assist you in troubleshooting many types of HPLC problems (to know which types of pressure changes indicate a real problem and which types of pressure changes are normal). Changes to the overall length or ID may result in noticeable changes to the total system backpressure. As an experienced chromatographer knows, when HPLC solvents are mixed together (e.g. gradient analysis) the pressure does NOT always follow a linear progression. In some cases, a reaction occurs between the solutions resulting in an overall change to the final viscosity of the mixture which may not be expected or understood by novice chromatographers (e.g. mixtures of MeOH/Water and ACN/Water are very well know examples which show these properties). 

 

You can download a free, more detailed table of 'HPLC Tubing Backpressure Examples' in PDF Format at this link:

The Three Most Common HPLC Questions and How To Solve Them

The three most common HPLC related questions I am asked each week can be summarized below. Test your basic chromatography knowledge. Before reading the answers, see if you can answer them correctly on your own.

• "What Is Causing the HPLC Baseline, Pressure or Peak Retention Time(s) To: Wander, Change, Drift, Vary or be Unstable?"

• "How Should I Wash or Regenerate My HPLC Column?"

• "How Can I Tell if the Sample is Retained On the HPLC Column? or What Does It Mean When No Chromatography Took Place?"

Let us address each question in order and attempt to provide accurate answers (I have included links after each question to articles with more detailed explanations).

What Is Causing the HPLC Baseline, Pressure or Peak Retention Time(s) To: Wander, Change, Drift, Vary or be Unstable?

• Retention times must be reproducible from run to run.The causes of an unstable baseline and/or changing peak retention time(s) are often related. Common reasons include: Column temperature fluctuations, inadequate mobile phase mixing or degassing, leaks, dirty column, sample overload, lack of pH or buffering control (weakly ionizable samples can be very sensitive to changes). *Full Article link with detailed answers, here.

How Should I Wash or Regenerate My HPLC Column?

Note: Before proceeding with any column regeneration or cleaning procedures, always refer to the specific advice provided by the column manufacturer. Approved maintenance and cleaning instructions can often be found in the product guide or booklet which comes with the new column. Additional information can be found on the vendor's website or by contacting them directly.

• Two issues must be addressed to answer these types of questions. (1) Always wash your column with a specific column wash solution which is stronger than your analysis solution. The use of a stronger solution (In this context, "stronger" means better at dissolving the samples and faster at eluting them from the column) as the wash solution requires regular use to maintain the column. Failure to regularly wash your column may result in compounds accumulating on the column over time (fouling the column) resulting in poor reproducibility, higher back-pressures, contamination and/or poor peak shape. (2) Next, always wash your column after each analysis. This should be a separate step, not incorporated into your analysis method. The analysis method should not include the column re-equilibration steps at all. A second, separate wash method should always follow each analysis method which includes the rinsing of the column with a "stronger" solution for an adequate period of time, then adjustment back to initial conditions where re-equilibration can take place to get it ready for the next analysis run. These are fundamental guidelines of good method development and follow well established principles. Developing methods in this way should increase the lifetime of your columns and improve the reproducibility of results obtained (better %RSD run-to-run).

For more information on washing bound proteins off RP HPLC columns, please refer to this linked article found here.

How Can I Tell if the Sample Is Retained On the HPLC Column? or What Does It Mean When the Sample Comes Out At or Near the Column Void Volume?

• Chromatography is a tool which when used properly adds one or more additional dimensions of physical or chemical characterization information to your analysis data. It does so first by using on-column RETENTION. Samples must be run under conditions which allow the material to interact with the chromatography support for a period of time. We define this time as the retention time. A sample which does not interact at all with the column support material will elute off the column early (and not be retained) at the "column void time" (or column dead time). We refer to this void time as the "T zero" time. When a sample elutes at or near the T zero time, no chromatography has taken place and no method has been developed. It is as if the HPLC column was not used. How do you know what the "T zero" time is (it will be different for different methods)? You must first calculate the HPLC column's dead volume. Once you know the column dead volume and flow rate, you can calculate the T zero time. A scientifically valid HPLC method will include conditions which retain the sample on the column for a long enough period of time to insure that it is interacting with the support. This allows for separation from other compounds to take place and is the purpose of chromatographic resolution. Without this retention mechanism, you are just flow-injecting the sample past the column and skipping all chromatography. It would be far simpler to just place the sample in a spectrophotomer cell as no retention or additional data would be obtained using that technique.

• When first learning liquid chromatography, two of the very first calculations you must learn to use in HPLC are: Column Dead Volume (aka: Column Void Volume) and the K prime of a sample (aka: Peak Capacity Factor). Do you know how to calculate these? They are calculated and reported for each method used. You should be able to tell anyone who asks you what the values are for each method. A chromatographer must know and understand them before using an HPLC system or running a method. They are also critical to method specificity and proper validation. Here are links which after reading and practicing, should make you an expert in these two fundamental calculations. 

"Determination of HPLC Column Dead Volume / Dead Time (T zero)";

"K Prime (also known as: Capacity Factor or Retention Factor): One of the Single Most Important HPLC Parameters of All".

So, how did you do answering these basic questions? If you have put in the needed study time and practical experience to learn and use these fundamentals of high-performance liquid chromatography, then you should have been able to easily provide correct answers to all three questions. If not, then it is time to go back and study up on those basic liquid chromatography texts and article links, plus get more supervised hands-on time with the instruments.

HPLC Retention Time Drift, Change, Area Variability or Poor Reproducibility. Common Reasons for it.

Retention times and area measurements must be reproducible from run to run. When problems are observed, late, early or variable retention times (and/or peak area values) may be observed. Variation outside of acceptable limits indicates a problem with the sample preparation, method design, function of the HPLC system or a lack of training. Here are several commonly observed reasons why sample (or standard) peak retention times or peak area values may not be reproducible:

(1) TEMPERATURE FLUCTUATIONS:

To obtain reproducible results, the temperature of the HPLC column must be kept constant or controlled during each analysis. Laboratory room temperatures can vary up and down by several degrees during the course of one day and these changes will often change the retention characteristics of the sample(s). The 'On' and 'Off' cycling of power from an air conditioner or heating unit will often cause the baseline to drift in a cyclical manner, up and down, during the day (this can often be seen as a clear sine wave pattern when you zoom-in to study the baseline trace over time). Temperature also changes the refractive index of the mobile phase. Optical (Light) based detectors (i.e. UV/VIS, RI...) will show this change as drift up or down). In some cases, a temperature change of plus or minus one degree C from run-to-run can cause changes in retention times which effect reliability of the method. 

To reduce temperature fluctuations, you must control the temperature of the column and mobile phase (if applicable) during the analysis. This is most commonly done by: (a) using fully equilibrated mobile phase at the start of the day or analysis, (b) keeping the interconnecting lines as short as possible (esp. any which exit the column and go to detectors/flow cells), (c) insulating any stainless steel lines with plastic tubing sleeves to reduce heat loss and (d) using a thermostatted column compartment to maintain the column at a single set temperature throughout the day. Control of the column temperature will remove 'temperature' as a variable from your analysis. Method analysis temperature should be constant from run to run, not a variable. Be sure and document the temperature selected as part of your method. 

(2) INADEQUATE MOBILE PHASE MIXING:

Both high pressure (with separate pumps) and low pressure pumping (one pump with a proportioning valve module) systems depend on efficient mixing to accurately meter the requested mobile phase composition. For gradient analysis, failure to completely mix the mobile phase solution before it enters the HPLC column often results in excessive baseline noise, spikes and poor Retention time reproducibility. If your mobile phase composition changes, then the chromatography will change too (e.g. evaporation of the more volatile organic phase from an open bottle may result in a change in composition). "Mixing" is often accomplished directly in a mixer installed in the flow path of an HPLC pump (For more info, please read this article on selecting a mixer). The associated noise and ripple of incomplete mixing can reduce the limit of detection (LOD) and increase integration error. This mixer is often a static mixer (a simple 'Tee', a tube filled with baffles, a frit or beads, valve orifice or microfluidic device) of low volume design for chromatography use, but allows adequate mixing of the liquids within a prescribed flow rate range. The best mixers incorporate longitudinal and radial mixing in-line. A mixer with too low a volume or of insufficient design can result in poor mixing of the mobile phase (note: incorrect solvent compressibility settings can also cause mixing, flow instability and noise problems too). To reduce mixing problems, first insure that the mobile phases used are fully soluble with each other. Next, make sure that any mixer used is appropriate for the flow rates and volumes you will be using. Monitor the baseline for drift, ripple and artifacts in real time to spot problems and make adjustments to correct them. 

(3) INADEQUATE MOBILE PHASE DEGASSING:

For the best results, continuously degass your mobile phase. Reducing the amount of gas in solution will also improve signal to noise levels of detection, reduce Retention time drift and reduce pump cavitation. If you are using an electronic vacuum degassing module, make sure it is maintained and working 100%. A faulty degasser may cause more damage to your system and methods. Maintain and Repair them just as you do for your other instrument modules. Gas bubbles may cause the inlet or outlet check valves to malfunction (get stuck), baseline noise spikes to appear randomly, flow rates and/or back pressure values to become irregular, detector outputs to show high levels of noise (from air in the flow cell) and also cause the loss of prime or cavitation in pumps. To achieve the best balance of low noise levels and high reliability, both aqueous and organic mobile phases should be fully degassed. This can be accomplished through stand alone vacuum degassing modules or through gentle helium gas sparging. In all cases, degassing must be continuous (not just done one time). Continuous degassing reduces cyclical noise and signal variations. For this reason, I do not recommend using ultrasonic baths to degass mobile phase solutions as these are not used continuously. The mobile phase solution starts to re-absorb gas as soon as you stop sonicating the solution. This results in continuous baseline drift.

Removal of gasses is critical to the function of a modern HPLC pumping system. The liquids used are compressed to very high levels which forces out solubilized gas from the solutions. This is best accomplished before the liquid is transferred into the pump. These gas bubbles must be minimized to achieve desirable baselines. *Even if you use a high pressure pumping system, an inline degassing system reduces the amount of noise and baseline drift. Properly maintain and service your degasser to insure compliant operation. IOW: Always degas your mobile phase solutions.

 

(4) SYSTEM LEAKS or FLOW RATE INSTABILITY:

If the peak retention times have increased over time, one possible reason for this change could be a leak. If your flow rate is reduced by a leak, then the retention times will be longer. Always be alert to this pattern of change and check for any signs of leaks on a regular basis. If you find a leak, do not use the HPLC system until it has been repaired. If there is no leak, then the flow rate may not be what you think it is. 

When the actual flow rate is in question, start by checking it manually (never trust the instrument's display screen or the software 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, measure the time it takes to fill a 10ml graduated cylinder. It must take exactly 10.00 minutes.

Inadequate degassing, sticking check valves and/or incorrect solvent compressibility values may also cause flow instability.

(5) COLUMN FOULING: 

One of the most common reasons for changes in retention times or area values of well established peak(s) are due to column contamination and fouling of the support material (or of the inlet frit, guard column). The most common reason for this to happen is due to a lack of column flushing or washing after each analysis (esp when running only isocratic methods). Samples that have been poorly prepared, not filtered or were sourced from a complex matrix (i.e. clinical samples) often contain many compounds which are in-addition to the compound of interest. These materials can be retained on the column and not eluted off during each analysis. They build up over time and cause all kinds of strange problems, including changing retention times, new peaks seen and poor overall or wide peak shapes. 

Gradient analysis provides an opportunity to make sure you use a strong enough mobile phase to elute everything off the column during the run. Make sure you ramp up to a high enough concentration of solvent and use a "hold time" to insure this.

Isocratic analysis is a worst case situation for this to occur as the mobile phase is not ramped up to a strong solvent at the end of the method to push off any late eluters, Instead, they accumulate on the column. 

If you use isocratic methods to analyze samples, then you must follow each analysis run with a second, and separate from your analysis method, "column only wash step". This method does not inject any sample. Instead, it uses a strong wash solution which is compatible with your column AND is well known to dissolve any accumulated material into solution and elute it all off the column. For NP applications an alcohol (e.g. MeOH) may be suitable for this job and for RP applications ACN or even MeCl may be be appropriate. Check with the column manufacturer to find out which wash solutions should be used (do not guess, base it on actual sample solubility). 

(6) SAMPLE OVERLOADING (or too large an Injection volume/concentration for the column): If you inject (load) more sample than the column can hold (as determined by a proper loading study), then the peak that results will often be broader in width with more tailing (from diffusion). This will result in a peak which elutes later than expected, fouls the column and results in poor reproducibility.  Be sure to inject the sample dissolved in the mobile phase (or a solution that is weaker than the mobile phase).

(7) SAMPLE INJECTION VOLUME VARIATION: The injection volume used must be appropriate for the type of injector used. All injectors have a stated range in which they are most accurate. Make sure you are injecting within this ideal range and not at the extreme ends of the range (larger error). Manual injectors with fixed loops should be overfilled (3x) for best results. Autosampler vials must be correctly chosen to be compatible with the injector used, contain an excess of liquid and have a loose cap to prevent evaporation or a vacuum from forming inside the vial. *Test injector accuracy and reproducibility separately, at the volume used for your analysis, as part of your method development review. *Review my article on HPLC injectors for more information.

(8) Changes in the pH OF the MOBILE PHASE: Samples containing ionizable compounds are strongly effected by the pH of your mobile phase. Solutions should be prepared fresh, each day (*acids in solvent may change over time). Buffer capacity is often overlooked (the ability to resist pH change). It is highest at the pKa of the acid/base. Try to work within ±1 pH unit of the buffers pKa value for the best pH control of the mobile phase. If your mobile phase is buffered too far away from its pKa, then poor peak shape or variable retention times are often the result. Note: Weakly ionizable samples can be very sensitive to changes of as little as 0.1 pH unit. 

Vacuum Pressure Units Conversion Table

Several of the questions I receive each week by email deal with scientific calculations or conversion of various units. One popular request relates to the conversion of micrograms, ppm and percent. Several years ago to address this question, I posted a table of weight to ppm units ("Conversion Factors microgram, nanogram, ppm, ppb and percent") which has proven to be very popular.

Because of the large number of vacuum pumps attached to HPLC and MS systems, another common conversion question relates to vacuum units. Due to the different applications and regions of the world, the desired unit often varies. It is for this reason that I develop unit conversion tables as I find these tables provide for a convenient way to print out and/or keep handy in a binder for future reference. Widespread computer use coupled to freely available page reader software (e.g. Adobe PDF) provides another means to store useful information as a pdf file too. I present this "Vacuum Pressure Units Conversion Table" in a viewable and an optionally available downloadable form [click HERE to download].

VACUUM PRESSURE UNITS CONVERSION TABLE:

*Some of the more commonly used values are shown in boldface type. ** Absolute Vacuum..

% Vacuum	Torr (mm Mercury)	kPa abs	Inches of Mercury	Micron	PSI
0.0	760.0	101.4	0.00	760,000	14.7
1.3	750.0	99.9	0.42	750,000	14.5
1.9	735.6	97.7	1.02	735,600	14.2
7.9	700.0	93.5	2.32	700,000	13.5
21.0	600.0	79.9	6.32	600,000	11.6
34.0	500.0	66.7	10.22	500,000	9.7
47.0	400.0	53.2	14.22	400,000	7.7
50.0	380.0	50.8	14.92	380,000	7.3
61.0	300.0	40	18.12	300,000	5.8
74.0	200.0	26.6	22.07	200,000	3.9
87.0	100.0	13.3	25.98	100,000	1.93
88.0	90.0	12	26.38	90,000	1.74
89.5	80.0	10.7	26.77	80,000	1.55
90.8	70.0	9.3	27.16	70,000	1.35
92.1	60.0	8	27.56	60,000	1.16
93.0	51.7	6.9	27.89	51,700	1.00
93.5	50.0	6.7	27.95	50,000	0.97
94.8	40.0	5.3	28.35	40,000	0.77
96.1	30.0	4	28.74	30,000	0.58
96.6	25.4	3.4	28.92	25,400	0.49
97.4	20.0	2.7	29.14	20,000	0.39
98.7	10.0	1.3	29.53	10,000	0.193
99.0	7.6	1.0	29.62	7,600	0.147
99.87	1.0	0.13	29.88	1,000	0.01934
99.90	0.75	0.1	29.89	750	0.0145
99.99	0.10	0.013	29.916	100	0.00193
99.999	0.01	0.0013	29.9196	10	0.000193
100	0.00	0	29.92	0	0