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).

Number of theoretical plates (N), Calculation Formulas

Number of theoretical plates (N):

Often used to quantify the efficiency (performance) of a column (HPLC or GC). "Plates" are expressed per meter of column length and should be calculated based on a retained peak with ideal peak shape or symmetry. 

   N = Plates; tr = Retention Time of Peak; w = Peak width;  w_0.5 = Peak width measured at half height.

Two popular formulas are:

Tangent: USP (United States Pharmacopeia / ASTM)

Best for Gaussian peaks. Peak width is often determined at 13.4% of the peak height (w). Inaccurate for peaks which are non-Gaussian, poorly resolved or tail.

   N = 16 (tr / w)²

Half Peak Height: (European Pharmacopeia)

For peaks which are less Gaussian in appearance, using a slightly different formula with the peak width measurement made at the half-height (W_0.5). Less accurate for peaks which are poorly resolved or tail.

   N = 5.54 (tr / w_0.5)²

 

• Other formulas, not included, for calculating Plate numbers include: Half Width, Variance Method, Area / Height & Exponential Modified Gaussian (EMG).
• Caution. HPLC column "Plate" values should not be used for a final determination of efficiency unless you are comparing all results on the same exact HPLC system, which is setup and run under identical conditions each time. Since the result is based on many possible variables, including how your HPLC system is plumbed (dwell volume & tubing ID), the peak's Kprime and symmetry, the detector used, sampling rate, integration quality, flow cell volume, flow rate, actual column used (to name a few), it can easily be manipulated to be very large or small.

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. 

HPLC K Prime. Also known as: Retention Factor, Capacity Factor): One of the Single Most Important HPLC Parameters of All

The role of Capacity Factor / Ratio (K prime) in liquid chromatography is to provide a calculation or ratio which defines how much interaction the solute (sample peak) has with the stationary phase material relative to the mobile phase (IOW: the relative time the sample spends interacting with the support vs. the mobile phase). If this interaction is too short (i.e. K prime less than 1.0), then no chromatography has taken place and you have just developed a "flow-injection" method (the same as if no column was used) instead of a chromatography method. The method fails all validation and is NOT fit for purpose. Sample Retention must be long enough to demonstrate that the method developed is specific to the sample and shows good selectivity (retention) for the sample analyzed. This is true for most, but not all modes of liquid chromatography (3). 

• New and ever 'experienced' users lacking training in HPLC often make this error, developing methods where the sample has little to no retention on the column. We routinely review methods where the actual K prime of the key sample is measured to be at or near 0, failing to show any chromatography took place in the method (*The Journals are filled with thousands of examples of invalid HPLC methods of analysis). This mistake is made because the author(s) have no formal training in liquid chromatography and do not understand the basics of the technique. 
• Note: Slowing down the flow rate to "show" a later elution time DOES NOT increase K prime (a very common novice mistake) !!! 
  

Observance of the fundamentals of chromatography are key to developing high quality HPLC methods. For most modes of HPLC, highest on this list of basic fundamentals is that the sample(s) be retained on the HPLC column used and not eluted out at or near the column's void volume (we often refer to this time in minutes as, "T-zero" or "t0"). Many chromatography methods fail this simple test of retention and are invalid as written. Knowing what a compound's retention or capacity factor is allows us to be confident that it has been retained and eluted past this critical point, but to first calculate K prime, we first need to know the HPLC column's void volume. 

Calculation and/or measurement of the Column Void Volume should be one of the very first chromatography method development tasks you learn to perform. Knowing the column void volume allows you to determine the retention time of an unretained sample and the resulting retention factor (K prime) of each sample eluted after it. To do this, you must calculate the column void volume AND inject a sample which will not be retained by the column to determine what time an unretained sample will be eluted off the column. This establishes the 'T' zero time, or T(0). The time it takes an unretained compound to elute off the column is critical to know. If your HPLC method does not retain the sample on the column long enough past this time, then you are not allowing any chromatography to occur. Once you have this T(0) value, you can then determine the retention factor (the "K Prime") of your actual sample(s) using the simple ratio formula below. Your final method should baseline separate all compounds apart and, if properly developed, each sample peak will often have K Prime values between 2.0 and 10.0. K prime values of greater than 10 are acceptable, but often show minimal improvements to resolution. Try and insure that the earliest eluting peak in your sample has a K Prime of  >1.5. Do not develop methods which only result in K Primes of less than 1.5 (an indication of poor quality chromatography). 

Note 1: Many regulatory agencies (e.g. The USA FDA) requires that K prime values for HPLC separations be equal to or greater than 2.0 to meet Specificity acceptance criteria (System Suitability/Method Validation). After all, if it elutes at or near the void volume, then your method is not specific for anything. Besides being unscientific in design, your method will fail System Suitability and fail validation. IOW: It does not meet this basic requirement.

• K Prime, K1 (Capacity Factor or Retention Factor) Formula:

       k1 = [T(R) - T(0)] / T(0)

• (where T(R) equals the retention time of the peak in minutes and T(0) is
  the retention time of an unretained peak). 
• *The 'K Prime' of your sample must be > 1.00. A value greater than 1.5 should be your goal.

Example #1: 
 T(0) found to be 2.90 minutes and the sample elutes at 5.80 minutes. k1 = 5.80 - 2.90 / 2.90. k1 = 1.00.

Example #2:
 T(0) found to be 2.90 minutes and the sample elutes at 9.10 minutes. k1 = 9.10 - 2.90 / 2.90. k1 = 2.13.

Example #3:
 T(0) found to be 1.75 minutes and the sample elutes at 1.74 minutes. k1 = 0. No retention and no chromatography have taken place at all. The method is invalid.


Note 2: I see and read published HPLC methods (including "Validated Methods" !) every week which ignore this fundamental requirement and present data showing little to no retention of the primary sample on the column. Most are RP methods run on popular C18 columns and show the main peak of interest eluting out as a nice sharp peak right at the void volume.  These methods often describe the sample analyzed as "100% pure" and are fully validated (because the person doing the work may not have had any HPLC experience or training)! A mixture will always look like a single peak by HPLC when no 'chromatography' is employed to separate out all of the possible components. The sample must be retained on the column for a period of time before we can conclude anything about its purity by the method employed.

Note 3: In some cases, when other modes of chromatography are utilized (e.g. ion exchange, size exclusion chromatography (SEC / GPC), K prime is not as relevant. The mode of chromatography can affect the interpretation. For example: This is because size exclusion chromatography relies on the sample's interaction with well defined pores inside the support (inclusion/exclusion) to separate based on molar size. A variety of pore sizes can be used to "filter" the sample. So a large K prime value might be normal for a molecule that is low in molecular weight and spends a lot of time working its way through the column. A high molecular weight sample might just "shoot" through the column due to little or no interaction with the pores. You still need to have retention on the column, but now it is determined by how long it takes the sample to find its way out of the column. SEC columns are bracketed by Pore Size (e.g Mw. Excluding all samples that do not "fit"). With size exclusion columns, determine the Retention and Exclusion times, not the K prime). This article is specific to thr more common cases where traditional HPLC NP or RP modes are used. In these cases, low K prime values indicate no retention took place and the method fails all claims of specificity for the sample (selectivity is absent or poor). HPLC methods with little to no selectivity fail scientifically as no chromatography has taken place.

CHIRAL HPLC / SFC METHOD DEVELOPMENT (Alcohols):

We are experts in chiral HPLC and SFC method development of pharmaceutical samples and have operated a contract separations labs for nearly two decades. We have learned a great deal about developing fast and reliable racemate separation methods, often where other companies have failed. The knowledge we have gained has allowed us to develop over ten thousand new chromatographic methods for our clients. This has made our company the leading expert in the chiral separations field. 

I would like to share a tip with you regarding the use of different alcohols in chiral method development. That tip is to experiment with different alcohols during the method development process (*Please make sure the alcohol is compatible with your column!). Many of the normal and reversed phase chiral columns can be used with some unconventional alcohols to achieve excellent separations. These alcohols are often used isocratically at 100% concentration for HPLC methods and at levels ~ 10 to 20% for many SFC methods. We have had a great deal of success using 100% pure Methanol for HPLC methods on normal phase style chiral columns (though 100% Ethanol is still one of the best alcohols to initially choose). For SFC methods, Methanol, Ethanol and Butanol (plus mixtures of these) are still some of our favorite co-solvents.Note: SFC needs these alcohols to make the compressed CO2 more polar. Since most chiral drug compounds are resolved using Normal Phase chromatography, SFC is still limited in what it can resolve chromatographically vs HPLC. SFC will never replace conventional HPLC as SFC is far more limited in application (restricted in its range of polarity), but SFC is a worthwhile and important technique to supplement HPLC separations. 

• Here is a list of some popular alcohols (HPLC grade) worth using in your chiral method development: 

Methanol; Ethanol;1-Butanol; 2-Propanol; 2-Butanol and Acetonitrile (I know this last one is not an alcohol, but it is often overlooked in chiral method development. It works where other solvent systems fail for both HPLC (100%) and less so for SFC (10% as a co-solvent with Methanol). 

Many of the above liquids can be used at 100% concentration, but others require mixing with Hexane or Heptane to yield lower concentrations of alcohol. *Remember to always consult with the column manufacturer first to determine which solvents are safe to use.

• A note about acids. Weaker is often better in chiral method development. Examples: TFA at 0.01% and Acetic or Formic Acids at 0.1% concentration are often strong enough to ionize most compounds.