What is Aqueous Normal Phase (ANP) and how can it help you?

Aqueous normal phase chromatography

From Wikipedia, the free encyclopedia

Aqueous normal phase chromatography (ANP) is a chromatographic technique which encompasses the mobile phase region between reversed-phase chromatography (RP) and organic normal phase chromatography (ONP).

In normal phase chromatography, the stationary phase is polar and the mobile phase is nonpolar. In reversed phase we have just the opposite; the stationary phase is nonpolar and the mobile phase is polar. Typical stationary phases for normal phase chromatography are silica or organic moieties with cyano and amino functional groups. For reversed phase, alkyl hydrocarbons are the preferred stationary phase; octadecyl (C18) is the most common stationary phase, but octyl (C8) and butyl (C4) are also used in some applications. The designations for the reversed phase materials refer to the length of the hydrocarbon chain.

In normal phase chromatography, the most nonpolar compounds elute first and the most polar compounds elute last. The mobile phase consists of a very nonpolar solvent like hexane or Heptane mixed with a slightly more polar solvent like isopropanol, ethyl acetate or chloroform. Retention increases as the amount of nonpolar solvent in the mobile phase increases. In reversed phase chromatography, the most polar compounds elute first with the most nonpolar compounds eluting last. The mobile phase is generally a binary mixture of water and a miscible polar organic solvent like methanol, acetonitrile or THF. Retention increases as the amount of the polar solvent (water) in the mobile phase increases. Normal phase chromatography, an adsorptive mechanism, is used for the analysis of solutes readily soluble in organic solvents, based on their polar differences such as amines, acids, metal complexes, etc. Reversed phase chromatography, a partition mechanism, is typically used for separations by non-polar differences.

The "hydride surfaces" distinguish the support material from other silica materials; most silica materials used for chromatography have a surface composed primarily of silanols (-Si-OH). In a "hydride surface" the terminal groups are primarily -Si-H. The hydride surface can also be functionalized with carboxylic acids [1] and long-chain alkyl groups[2]. Mobile phases for ANPC are based on an organic solvent (such as methanol or acetonitrile) with a small amount of water; thus, the mobile phase is both "aqueous" (water is present) and "normal" (less polar than the stationary phase). Thus, polar solutes (such as acids and amines) are most strongly retained, with retention decreasing as the amount of water in the mobile phase increases.

Typically the amount of the nonpolar component in the mobile phase must be 60% or greater with the exact point of increased retention depending on the solute and the organic component of the mobile phase. A true ANP stationary phase will be able to function in both the reversed phase and normal phase modes with only the amount of water in the eluent varying. Thus a continuum of solvents can be used from 100% aqueous to pure organic. ANP retention has been demonstrated for a variety of polar compounds on the hydride based stationary phases (J.J. Pesek, M.T. Matyska, J. Sep. Sci., in press).

An interesting feature of these phases is that both polar and nonpolar compounds can be retained over some range of mobile phase composition (organic/aqueous) as a result of residual silanol groups acting in a HILIC mode. This property distinguishes it from a pure HILIC (hydrophilic interaction chromatography) column where separation by polar differences is obtained, or a pure RP stationary phase on which separation by nonpolar differences in solutes is obtained with very limited secondary mechanisms operating.

Another important feature of the hydride-based phases is that for many analyses it is usually not necessary to use a high pH mobile phase to analyze polar compounds such as bases. The aqueous component of the mobile phase usually contains from 0.1 to 0.5% formic or acetic acid, which is compatible with detector techniques that include mass spectral analysis

1. What is “Aqueous-Normal Phase” Chromatography?In Aqueous-Normal Phase, the maximum retention time of analyzed compounds is when 100% acetonitrile (least polar solvent) is used as the mobile phase and as you increase the polar solvent content (Aqueous), retention is decreased.

2. What is an Octanol/Water Partition Coefficient?Basically, this is a measure of the hydrophobicity v. hydrophilicity of a compound. It is extremely useful when combined with the pI of your molecule to predict retention times.

The Octanol-Water Partition Coefficient is a physical property used to describe a chemical’s lipophilic or hydrophobic properties. It is the ratio of the concentration of your compound in the octanol phase to its concentration in the aqueous phase at equilibrium. It is commonly measured and labeled as Log P. Compounds with large non polar structures usually have high logP values and for compounds with highly polar groups, it is usually very low.

3. What is a Ballistic Gradient?A ballistic gradient is a very fast separation technique used mostly in LC-MS applications; the complete analysis can take less than one minute and up to five. Non-optimal, high flow rates or linear velocity are combined with fast gradients and very short columns.

4. What can cause excessive back pressure?There can be many causes of excessive back pressure but one very common cause is build up of foreign materials on the top of the HPLC Column. This situation can result when sample precipitates (retains or accumulates) or dust and other particles from the degradation of the pump seals.

This situation can be avoided by the use of a guard column; when you reach excessive back pressure, simply replace the guard column.

5. When should I use an Amino Column?Amino columns are used mainly in Normal Phase HPLC for separation of polar compounds which are difficult to retain and separate by RP-HPLC. The main class of compounds which are separated using amino columns are: oligosaccharides, glycoalcaloids, surfactants, polar pharmaceuticals and impurities, tocopherols.

Cogent Type C columns can be used to separate the above compounds in Aqueous Normal Phase mode using acetonitrile / DI water mobile phases.

6. What is Dwell Volume?Dwell volume is simply the time delay for a gradient change to reach the top of the HPLC column . It is important to remember that each HPLC system has its own dwell volume and will effect the separation results. THIS IS A MAJOR REASON GRADIENT METHODS MAY NOT TRANSFER. Click here to view how to Determine Dwell volume.

7. What are the advantages of using a 3.0mm ID HPLC column for LCMS?Many scientists use a 2.1mm ID and smaller columns for LCMS which have optimal flow rates of approximately 0.3ml/min. This is fine for isocratic analysis but when you must use a gradient method, specialized pumps and mixing valves are necessary that can reproduce the very small changes in the mobile phase’s composition. 3.0mm ID columns use more

traditional flow rates and can be used easily on LCMS without specialized pumps for both gradient and isocratic methods. Also, when using small column ID’s it is important that you invest the time to completely optimize your system’s plumbing to minimize extra-column band broadening to achieve acceptable chromatography. If you have sufficient sample for loading, you may want to consider using a 3.0mm ID column for any situation that a 2.1mm ID has been recommended for all of these and other reasons.

8. What determines the retention or hydrophobicity of a molecule in HPLC?Much of the retention of a column in Reverse Phase chromatography is due to what is commonly called hydrophobicity. This natural phenomena is due mostly to the size of the hydrophobic (water resistant) area of a molecule. Retention will increase with the amount of water in the mobile phase. In general, the more hydrophobic the molecule (see Octanol/Water Partition Coefficient) the longer it should be retained.

9. What life time should I expect from a Cyano Column?While Cyano columns have suffered from a reputation of not lasting very long, with type B silica (high purity) and proper bonding technologies, a Cyano column that is conditioned and treated properly should last as long as any other column. To lengthen column life I recommend that you avoid using mobile phases with a pH higher than 7.0 with Cyano and Amino columns.

10. Why does the metal content of a silica particle contribute to the peak shape of my chromatogram?The electron withdrawing properties of trace metals in silica particles enhances the negativity of surface silanol groups and contributes to peak tailing in silicas with high levels of trace metals.

11. Should I filter my HPLC Solvents and Buffers before use?The most common cause of excessive back pressure is due to the accumulation of particulates and permanently adsorbed materials at the top of the HPLC Column. This could be precipitate or un-dissolved particles which can come from your samples or your mobile phase. The best way to minimize accumulation on your column is to filter your samples, solvents and buffers with a .45µm or a 0.2µm syringe filter or through a filter flask before use. This should give you more column lifetime. Another way to minimize this build up is to use either an inline solvent/mobile phase filters a guard column or a MicroSolv Column Saver. These disposable devices are low cost and easy to use without disrupting your chromatographic results.

12. Why do some methods use low pH buffers to separate acidic compounds?By suppressing their ionization at low pH, acids will be more like neutral compounds and will retain on HPLC columns much the same way that neutral compounds will. Another benefit of running on Type A and Type B silica at low pH is that the silanol activity is reduced. This should sharpen your peaks.

13. I am confused when it comes time to use Phenyl, Amino or Cyano Columns. What are the main differences?All these columns will offer different selectivities in reverse phase compared to a C18 or C8 column. Amino, and Cyano columns can be used as both normal and reverse phase; it is best to dedicate a particular column for either reverse or normal phase and not to change back and forth. Phenyl columns are more robust and rugged than cyano and amino but normally used only in reverse phase separations. Phenyl columns like C18 or C8 tend to retain samples

based on hydrophobicity. A mixed interaction involving hydrogen bonding and dipolar interactions are responsible for the retention of solutes on cyano-silica columns and weak hydrogen bonding interaction leads to separation on amino-silica columns.

Note: Cogent Type C columns can be used in both Reversed and Normal Phase modes. A simple 30 minute procedure allows switching from one mode to another.

Procedure:

A – for moving from RP-HPLC to NP-HPLC pump 100% methanol for 15 minutes at 1 mL/min. flow rate, followed by 15 minutes 100% methylene chloride. Column is ready to be equilibrated with mobile phase for NP-HPLC.

B – for moving from NP-HPLC to RP-HPLC pump 100% methylene chloride for 15 minutes at 1 mL/min. flow rate, followed by 15 minutes 100% methanol. Column is ready to be equilibrated with mobile phase for RP-HPLC.

14. What is the first step that you would recommend in developing a new HPLC method and where should I start?An easy way to start any HPLC method development is to look in the USP, BP or other reference sources. If you cannot find a similar method to modify, contact one of the HPLC column manufacturers. Often their technical support can be very helpful and at no cost. See our offer for method development help on our website.

15. What performance standards should I set for a routine method? How do I go about developing this?You should define your goals as loosely as possible then determine resolution, tailing, precision, LOQ and linearity goals. Also include such important factors as maximum analysis time, sample prep complexity factors and cost. You may want to also develop your methods to be LCMS compatible.

16. I heard that rapid changes in column back pressure can damage the column. Is this true?Yes, definitely. Exposure to rapid changes in back pressures in HPLC columns can cause a type of “pressure shock” and cause damage. Limit the pressure in your columns to 5,000 psi and avoid rapid changes such as manual injectors that “slam” the pressure. For this and other reasons, it is best to operate your HPLC system with pressure cut off around 3,500 psi. This will protect your columns from this damage.

17. What special care should I take to protect my columns when I am equilibrating them?When you are equilibrating your columns for the first time or whenever changing your HPLC methods it is wise to consider not putting anything into the columns that can precipitate or is immiscible with the storage or current solvent in the column. Always ensure that the column is fully equilibrated with your mobile phase before making any injections.

Depending on column type, this could range from 10 to 15 column bed volumes (click here to view our instructions on line) for typical reverse phase to 50 to 100 bed volumes with polar embedded phases.

Beware of buffers that might be in your column before you change the mobile phase composition with respect to the organic content. This can cause immediate precipitation and ruin your columns.

18. What are the typical flow rates recommended by HPLC column companies to start with, for various column ID’s?Although it will vary from column maker to column maker, and is very dependant on the column length back pressure the following is good rule of thumb for common columns.

Column Dimensions

Recommended Flow Rate

1.0 30-60µL/min

2.1 0.1-0.6mL/min

3.0 0.3-1.5mL/min

4.6 0.8-3.0ml/min

7.8 4.0-10mL/min

19. What would you recommend to reduce time to develop an HPLC method?This is simple. Use short columns, increase flow rate to 2-2.5mls/minute and find appropriate bonded or stationary phases to match your mobile phases and solubility of your analytes. The easiest way to accomplish the latter is using out Mini-Scout™ Strategy.

20. What is a "system peak"?Both analytical and preparative liquid chromatography separations are often performed using mobile phases containing more than one component. When samples are dissolved in a different solvent than the mobile phase, after the injection an additional signal called the “system peak” can appear. The presence of these peaks is explained through loss of equilibrium in the analytical or preparative column caused by competitive interactions between the separated solutes and the strong additive of the mobile phase. During the relaxation process the system peaks are being generated. It is worth noting that even if the system peaks are often misinterpreted, they offer valuable information regarding the thermodynamics and kinetics of the separation, which takes place in the chromatographic system.

However from the method development point of view system peaks should be avoided by dissolving the sample in a solvent that closely matches the mobile phase composition.

21. I am analyzing metformin in plasma samples using acetonitrile + DI water with 0.5% formic acid. How I can obtain reproducible results and peak shape?You can try substitute formic acid in DI water by adding ammonium acetate (10 mM) and acetic acid (1%) to the aqueous phase. This should improved the reproducibility of peak areas and retention times. In addition extensive reconditioning of the analytical column with pure acetonitrile in between separations is essential for good reproducibility when working with plasma samples.

22. What is the maximum changes I can make to a method to increase the chances of meeting system suitability in HPLC?This is a question that is always asked and the following are only guidelines. For exact details, consult the FDA or the USP.

Change of Detector Wavelength

Not Allowed (you can vary up to 3nm in accuracy but you cannot adjust the detector.)

Column ID +/- 50%

Column Length +/- 70%

Column Temperature +/- 20%

Concentration of buffer salts

+/- 10%

Flow Rate +/- 50%

Particle Size - 50%

pH of Mobile Phase +/-0.2 units

Ratio of Mobile Phase Solvents

+/-10% absolute (This adjustment is not suggested)

23. Can I use trifluoroacetic acid (TFA) to improve the chromatographic performance in LC-MS analysis?Trifluoroacetic acid (TFA) is very often used as an additive in HPLC, because of its excellent solvating and ion pairing characteristics. It is a highly volatile acid as well, which could make it an additive for LC-MS analysis. However in many studies it was found that TFA causes spray instability and ion suppression in APCI (atmospheric-pressure chemical ionization) and ESI (electro-spray ionization) ionization techniques. In a recent study 1 it was found that TFA was the worst additive for ESI or APCI and formic acid was the best choice.

Note: Regardless of the additive used in LC-MS the response of analyte decreases with increasing concentration of ionizing agent. For this reason it is important to keep the concentration of an additive as low as possible.

1 D. Temesi, B. Law, LCGC 17(7), 626 (1999).

24. Since Cogent TYPE-C™ columns can be used in Reverse Phase or Normal Phase modes, what do you suggest to “switch” them since these solvent systems are not miscible? Just as a reminder, all Cogent Bidendate C18 and C8 columns are filled with water containing solvents during shipping. To start work in Normal Phase Mode, a simple switching procedure is recommended.

A simple 30 minute procedure allows switching from one mode to another.

Procedure:

A – moving from Reverse Phase to Normal Phase HPLC; pump 100% methanol for 15 minutes at 1 mL/min. flow rate, followed by 15 minutes 100% methylene chloride. The column is ready to be equilibrated with mobile phase for NP-HPLC.

B – moving from Normal Phase to Reverse Phase HPLC; pump 100% methylene chloride for 15 minutes at 1 mL/min. flow rate, followed by 15 minutes 100% methanol. The column is ready to be equilibrated with mobile phase for RP-HPLC.

25. When I run gradients on my HPLC and switch from one gradient to another, my baseline shows as “negative”. If I auto zero, it becomes positive for the remainder of the run but when I start again, it is negative. What can you suggest?Negative baselines in gradients are not that unusual. If the method is using even moderately UV absorbing components at the wavelength of interest, it is very difficult to exactly balance the absorbance signals of both channels. Acetic acid is very bad in this respect. The lower the wavelength, the more difficult it is to manage. Getting a smooth gradient with peptides at 214nm and TFA is an excellent example of the challenge at hand.

The only suggestion we can have for you is to start with fresh mobile phase and to make sure that your reagents and solvents are as pure as you can afford. Sometimes columns will retain these impurities from the solvents and reagents and will release during a run. If you observe this, I suggest you also use a fresh column.

Frequently Asked Questions

1. Can a Kromasil AmyCoat or CelluCoat chiral column be used in both Normal Phase and Reversed Phase mode?

Yes, the columns can be operated under normal phase, polar mode and reversed phase conditions. The regular columns are packed to give highest possible column efficiency under normal phase and polar mode conditions (alcohol or acetonitrile, no alkane). They can be converted to reversed phase via 100% ethanol or isopropanol as an intermediate solvent. Of course, the columns can also be re-conditioned to the original mode. By converting the column, one will observe a loss in column efficiency when comparing the plate count in RP vs. NP mode. For regular use under RP conditions, we recommend the purchase of columns that are packed giving highest possible efficiency under reversed phase conditions, AmyCoat RP and CelluCoat RP.

2. Can the column be operated with 100% aqueous mobile phase?

The reversed phase Kromasil columns C4, C8 and C18 should be operated with at least 5% organic modifier. Under 100% aqueous conditions, the columns are likely to undergo dewetting which means the mobile phase will be expelled from the porous system due to surface tension. In this case the analytes elute more or less with the void volume. The problem arises mainly if the flow is stopped. If a column has been dewetted, it can easily be restored by pumping mobile phase with at least 50% organic modifier through the column for ca 40 min.

Kromasil Phenyl, Kromasil Eternity phases (C18 and PhenylHexyl) can be used under 100% aqueous conditions without risk for dewetting.

3. How can column lifetime be maximized?

Column plugging is responsible for well over half of all column failures. Therefore thorough filtration (0.2 μm) of mobile phases and samples is a key for long column lifetime.

The mobile phase compatibility is another important factor. Standard RP stationary phases are stable under pH 1.5 to 9. Under certain conditions (organic buffers) the stationary phases are even chemically stable up to pH 12. Please consult with our technical support if you would like to use your classic Kromasil column at pH values exceeding pH 9. Generally, organic buffers are milder towards the silica based stationary phase than inorganic ones, such as phosphates or carbonates. If you need to run your method at high pH choose Kromasil Eternity which is stable up to pH 12.

Further, the columns should never be left in a buffered mobile phase when the flow is interrupted for more than 10 minutes. Either choose a very low flow rate (0.1 ml/min for a 4.6 mm ID column) instead of stopping the flow completely, or exchange any buffered mobile phase with non-buffered one (e.g. water/acetonitrile 70/30). This will prevent the column from being clogged with precipitated buffer salts.

4. How can the column performance be checked?

The performance is best checked by re-running the separation shown on the enclosed test chromatogram. The exact conditions are mentioned. The sample amount should not exceed the following quantities:

Column Ø [mm ID]

test sample

[µg]2.1 24.6 1010 5021.2 225

In order to achieve representative plate counts, it is important to reduce the extra column volumes, e.g. correct tubing has to be installed between injection and the column and from the column to the detector.

5. How can the columns be cleaned / regenerated?

An increased back-pressure, altered retention times and loss of column performance are all symptoms of deposits in the column or on the surface of the stationary phase. Most of the times, these problems can be overcome by the use of a correctly applied washing procedure. What should also be kept in mind is that in most cases the sooner washing (regeneration) of the column is performed, the better.

Strongly adsorbed species are collected at the solvent-inlet end of the column and in many cases it is a benefit to use a reversed flow during washing. Worth mentioning is that a well packed column should not loose performance as a result of reversed flow.

RP (Hydrophobic Phases)

Deposits are most commonly present as surface adsorbed species or precipitations.

Examples of recommended solvents

Suspected Deposition/ImpurityRecommended solvents

Examples

LipohilicStrongly Lipophilic solvents

Al, Tol

Polar (Small peptides) Versatile solvents DCM, THF, DMFStrongly Polar / Ionic (water sol.)

Aqueous solvents 50:50 DMF/w, THF/w

Polar, positively charged (amines)

Ion-exchange supressing mixtures

DMF/AA (1%), DMF/TFA (0.1%)

Macromolecular Depositions (Protein/ Large Peptide precipitations)

Strongly interaction breaking mixtures

DMF/1% SDS(aq), ACN/1% SDS(aq), Alc/AA, Alc/NEt3 (0.1%) Alc/10-100mM aqueous NaOH*

Salt/Buffer precipitations

Highly aqueous mixtures

Alc (10%)/w

  Al = Alkanes, Tol = Toluene,

Examples of recommended solvents

Suspected Deposition/ImpurityRecommended solvents

Examples

DCM = Dichloromethane, THF = Tetrahydrofuran, DMF = Dimethylformamide, AA = Acetic acid, TFA = Trifluoroacetic acid, ACN = Acetonitrile, Alc = Alcohol (MeOH, EtOH), w = water, NEt3 = Triethylamine, SDS = Sodiumdecylsulphate

* Use as last measure for not more than 10 column volumes, also end by acidifying the phase sing Alc/1% aqueous AA 50:50.

Regeneration protocol

1. Choose solvent/solvent mixture based on impurity. If not known, assume strongly polar / ionic species in order to avoid further precipitation.

2. Use a low flow rate (10% of normal flow rate), possibly in reversed flow mode and at slightly elevated temperatures (< 40°C), for up to ten column volumes.

3. If problems with back-pressure, occasionally check progress at normal flow.4. If the problem remains apply conditions for polar, positively charged and/or

macromolecular depositions. If SDS is used, wash thoroughly with pure THF, DMF or ACN afterwards.

5. If problems persist please consult our technical support for further advice.

NP (Polar Phases)

For polar phases, especially bare silica, very strong adsorption of polar residues is common. While under normal phase conditions highly polar impurities are not eluted, however the solution to this is the use of high levels of protic solvents, possibly in combination with acid.

If there are problems with altered retention times, first check the phase - water (moisture) equilibrium. The use of dry solvents could gradually "dry" the stationary phase, resulting in altered retention times. This is especially rue for bare silica.

Regeneration protocol?

1. Eqilibrate the column using an aqueous-lipophilic mediating solvent such as THF.

2. Use a low flow rate (10% normal flow rate) of pure Alcohol (MeOH), possibly in combination with acid (Acetic acid or Formic acid, 1-5%), for up to ten column volumes.

3. If the problem persists, use a mixture of Alcohol and water, with added acid, under the same conditions as in 2.

4. As a final measure: 0.5-1.5% NH3(aq), followed by 0.5-1.5% aqueous acid (HCl, AA etc.), water, alcohol, THF and finally the actual eluent.

5. If problems persist please consult our technical support for further advice.

?: While using highly polar solvents, especially for bare silica, the stabilization of the system after washing could take a significant amount of time. This is due to the silica surface - water equilibrium being distorted.

6. How should a buffered mobile phase be prepared?

Buffered mobile phases can basically be prepared in two ways:

1. preparing the buffer, including pH adjustment prior to mixing with organic modifier.

2. mixing buffer and organic modifier prior to pH adjustment.

While the pH is a well defined parameter in aqueous systems, it is not as straight forward to define or measure proton concentration in partially organic solvent systems.

It is therefore recommended to prepare the aqueous buffer, pH adjust it, filtering it through 0.4 µm and mixing it thereafter with the organic modifier. Attention should be given to the following points:

buffers are effective within +/- 1 pH unit from their pKA ? choose correct buffer system

consider the risk of precipitation if high buffer concentrations are mixed with organic solvents

always filter buffers through 0.4 mm filter prior to use be aware of the risk for bacterial or fungal growth in pure aqueous buffers the pH generally shifts upon the addition of an organic modifier (pH increases

for inorganic buffers (e.g. phosphates) and decreases for organic buffers (e.g. acetates) Example: phosphate buffer pH 7.6 ? addition of 40% MeOH ? pH 8.5

7. How should the columns be stored?

Correct column storage is a prerequisite for problem free chromatography and a long column life time. Normal phase columns should be stored in heptane/2-propanol or another inert solvent that is free from additives.

Reversed phase stationary phases should be kept in a mixture of water / organic modifier, free from any buffers or additives that might precipitate during storage. Thus it is recommended to wash the column whenever chromatography is going to be stopped for more than 15 min with water / organic modifier 50/50, independently on whether the column will be detached from the HPLC instrument or not. If chromatography is only to be interrupted for a couple of hours it might be less laborious to keep the buffer containing mobile phase running at a very low flow rate. If the column is detached, close the inlet and outlet with a screw in order to avoid the stationary phase to dry out.

All HPLC columns should be stored at ambient temperature (18-26°C), without risk for mechanical shock, preferably in the original column box.

8. What can cause poorly reproducible retention times?

1. Mobile phase composition

Most reproducible results are obtained by weighing the mobile phase mixtures. For water / ethanol 30/70 (v/v) 300.0 g (300 ml x 1.0 g/ml) water are mixed with 546.0 g (700 ml x 0.78 g/ml) ethanol.

Especially when working with alcohols as polar modifier, online mixing by the HPLC pumps should be avoided, as the viscosity of the components varies significantly and hinders proper blending.

Mobile phases should always be prepared fresh, as evaporation will alter the composition of a two component mobile phase.

2. Shifting temperature

A difference of 5°C can render +/- 10% shift in retention times. It is therefore recommended to work with a column oven even when working under ambient temperature.

3. pH Control

The retention time of acidic and basic analytes depends upon the pH value. 0.1 pH unit can render a shift in retention time by 10%. It is therefore recommended to work at a pH value at least 2 pH units from the pKA.

4. Incomplete equilibration

It is recommended to equilibrate reversed phase columns with at least 10 column volumes, for buffered mobile phases with at least 20 column volumes before an analysis is initiated. Make sure all mobile phase channels are purged with the adequate mobile phase. Normal phase columns can require up to 10x longer equilibration times than reversed phase columns. Bare silica columns might take hours to equilibrate, special attention should be given to the water content in the normal phase systems. Most reproducible results are often obtained when deliberately adding 0.05% water the mobile phase.

5. Dewetting

Highly polar mobile phases can be expelled from the hydrophobic porous system, leading to less and less accessible surface area, and therewith causing shorter and shorter retention times. See also: "Can the column be operated with 100% aqueous mobile phase".

9. What does end capping mean?

End capping describes the process of reacting silanol groups that have not been derivatized, e.g. octadecylchlorosilane. The end capping reagent is generally a smaller silane (e.g. trimethylchlorosilane) that is used to react with some of the residual hydroxyl groups. This treatment reduces the amount of available silanol groups that can undergo often undesired interactions with polar or charged analytes.

10. What does HILIC mean?

HILIC means Hydrophilic Interaction Liquid chromatography and is a technique used for separating highly polar substances. It can also be described as aqueous normal phase chromatography. The stationary phase has to be of polar character, e.g. silica, diol, amine, amide or zwitterionic. As a mobile phase, generally acetonitrile / water mixtures are used, where water is the strong component, thus exhibiting a higher elution strength than acetonitrile. Thus, HILIC is a sub-category of normal phase chromatography and shows opposite elution pattern to reversed phase.

11. What is "phase collapse / wettability" problem?

The use of mobile phases with a low content of organic modifier (< 5 %) generally leads to what is known as "phase collapse". The latter is best described as a dewetting phenomenon, in which the highly aqueous mobile phase is excluded from the hydrophobic pore system due to surface tension. The effect is observed predominantly with reversed phase packing materials with high ligand density (> 3.2 μmol/m2). The loss of wetted surface results in a decrease in accessible interaction sites for the solute, and leads to a loss in retention and / or reduced loading capacity. The process can easily be reversed by purging the packed bed with a high content of organic modifier (> 50%) The phenomenon is most dominant for C18 and C8 packing materials with pores < 150 Å, but even with C4 modified packing materials, 100% aqueous mobile phases should be avoided in order to assure robust chromatography.

12. What is an appropriate flow rate for a certain column?

For each particle size, there is an optimal linear flow rate with respect to column efficiency, according to the van Deemter plot.

Optimal flow rate (ml/min) for Kromasil columns depending on particle size and column diameter.

  3.5 µm 5 µm 10 µm 13 µm 16 µmLinear flow rate (cm/min)

7.1 4.7 3.3 2.4 1.9

2.1 mm ID 0.3 0.2 - - -4.6 mm ID 1.5 1.0 0.7 0.5 0.410 mm ID - 4.7 3.7 2.4 1.921.2 mm ID - 21 15 10 8.5

For shorter separation times, the flow rate can however be increased until the pressure drop limit of the HPLC pump is reached, e.g. ca 400 bar.

13. What is the difference between dead volume and dwell volume?

The dead volume can also be called extra column volume and consists of the volume of a HPLC system between the point of injection to the point of detection, but excluding the column itself. Thus it includes the injection volume, the volume of the injector, the volume of the connecting tubing before and after the column, the volume of the end-fittings and frits, and the volume of the detector flow cell. The dead volume can be measured by replacing the column with a zero-volume connector. By injecting a very small sample amount, the time can be measured between the moment of injection and the maximum peak height. This time multiplied by the flow rate gives you a good estimate for the system dead volume.

The dwell volume or gradient dwell volume is responsible for the time delay of the gradient. It describes the volume of a gradient HPLC system between the mixing chamber and the column inlet. This volume does of course also exist under isocratic elution, but in that case, it has no impact on the separation. The gradient dwell volume comprises of the volume of the gradient mixer, the connecting tubing to the pump, the pump head and check-valves, the tubing between the pump and the injector, the injector itself, and the tubing between the injector and the column inlet. When initiating a gradient, the column is not subjected to the change in eluent composition until the gradient has passed the dwell volume. During that time, the column is operated under isocratic elution. Attention has to be given when transferring a gradient method from one instrument to another. If the dwell volume differs, the retention times will likely differ as well, despite an identical method and column. The dwell volume can be measured by running a step gradient from 100% methanol to 100% methanol + 10 mg/L acetophenon. The UV detector will detect an S-shaped detector trace. The dwell volume is equal to the time between the injection and half height of the detector trace, multiplied by the flow rate.

14. What is the difference between Reversed Phase (RP) HPLC and hydrophobic interaction chromatography (HIC)?

In both cases, the stationary phase is more hydrophobic than the mobile phase. The hydrophobicity of RP-stationary phases is generally higher than that of HIC media. Elution in RP is obtained by adjusting the polarity of the mobile phase with a water miscible solvent, e.g. an alcohol or acetonitrile. Elution takes place either under isocratic (constant modifier concentration) or a under gradient conditions (increase in modifier concentration).

HIC media has weaker hydrophobic character and elution is induced by decreasing the polarity in the mobile phase by virtue of a decrease in salt concentration. HIC is generally applied in protein separations, where exposure to organic solvents under prolonged time might induce irreversible unfolding, thus loss of biological activity of the proteins.

For analytical purposes, RP-HPLC is an unchallenged method when it comes to separating closely related substances, including proteins.

15. What is the pressure drop limit for your chiral stationary phases (CSPs)?

There is virtually none! Both the polysaccharide based CSPs Kromasil AmyCoat and CelluCoat, as well as the CSPs based on a crosslinked network polymer, Kromasil

TBB and DMB, are based on highly mechanically stable, in-house produced, porous silica spheres. Moreover the proprietary coating method for the polysaccharide based phases makes these CSPs highly resistant to hydrodynamic forces. Thus, all Kromasil CSPs can be operated up to 400 bar back pressure. Exploiting the full pressure drop range of classical HPLC instruments allows for fast analysis and moreover very efficient column equilibration at high flow rates.

16. What is the shipping solvent?

The shipping solvent is stated on the test chromatogram and depends upon the surface modification of the stationary phase.

17. What should be done if the back pressure is rising?

When pumping mobile phase through a particular bed, a certain pressure drop δP over the column will result. The relationship between particle diameter dp and the flow velocity u is given by the Darcy´s law for non-compressible solvents:

Accordingly, δP is proportional to the linear velocity u, the column lengths L and the viscosity of the mobile phase η, and inversely proportional to the constant k0 and the square of the particle diameter dp. The constant k0 is a function of the interstitial porosity εi according to the Karman-Cozeny equation:

The equation above shows that the interstitial porosity εi has a tremendous influence on the permeability of the bed and therewith also on the pressure drop. For a well packed reversed phase be the interstitial porosity can be assumed to be 0.33 which leads to k0 = 4.45.10-4. The column permeability (d p

2·k0) does in practice depend entirely upon the particle diameter and the particle size distribution. The latter is a very important parameter, since the particles of an HPLC packing material are never mono-disperse.

Broad distribution has two negative effects:

1. the largest particles control the efficiency and2. the smallest particles control the pressure drop.

Hence, broad particle size distribution should be avoided.

18. What type of tubing/capillaries and connectors should be used?

Capillary tubing

When working with analytical HPLC instruments, capillaries with external diameter of 1/16" (1.6 mm) are generally used to connect the different parts of the instrument. Those parts that do not affect the extra column volume should have internal diameters (I.D.) of 1 mm. They bring along little risk for clogging and show minimum flow resistance, which contributes to the extra column pressure drop. For those connections critical for the extra column volume, internal diameter of 0.17 mm is recommended. Such capillaries add only 23 μL extra volume per meter of tubing (1.0 mm ID tubing: 800 μL/m). In order to avoid clogging of such fine capillaries, it is of uppermost importance to filter both, mobile phases and sample solutions.

For preparative columns the tubing has to be scaled up in order to reduce the flow resistance caused by the higher flow rates (δPcap≈dcap

-4):

Tubing sizes for column sizesColumn I.D.

mmTubing I.D.

mm20 0.2550 0.5100 1.0

Fittings

Fittings are connecting the various parts of the HPLC instrument. Steel fitting usually have a separate ferrule, some designs even use two ferrules, a ring and a conical piece. When tightened the first time, the ferrule is pinched permanently and the surrounded capillary is slightly compressed. The ferrule can no longer be removed from capillary. Plastic (PEEK) fittings are one single piece (nut, ferrule), and are installed by finger tightening only. Over tightening (common with steel fittings) must be avoided, since excessive force is likely to damage the threading and the capillary. It is recommended to tighten the screw only slightly with a wrench and to test if the connection is pressure resistance, meaning, no leakage occurs once mobile phase is pumped through the system.

The distance from end of tubing to ferrule lowermost end is depends upon the column and/or instrument design. Some examples are given below:

0.130" in case of Waters columns 0.80" for Valco design 0.170" for Rheodyne injector design

19. When is a guard column recommended?

A guard column can be useful if the injection sample consists of a complex matrix with partially unknown content. However, a guard column does never make sample filtration redundant! If a guard column is to provide adequate protection for the analytical column, it must be changed often enough in order to avoid the column performance from deteriorating. By monitoring plate number (N), pressure, and resolution (Rs), the performance of the guard column, as well as the analytical

column, can be assessed and a suitable moment for exchanging the guard column can be chosen.

As a rule of thumb, the guard column should be replaced when one of the following points applies:

N decreases by > 10% pressure drop increases by > 10% Rs changes by > 10% more than 150 samples are injected.

20. When should a gradient be used?

In reverse phase HPLC it is recommended to run a scouting gradient if the conditions for a successful separation are unknown. Such a run is performed from ca 10-80% organic modifier during 30-50 min, using a linear increase in elution strength. Based on the result of this scouting gradient, either an appropriate mobile phase composition can be chosen for isocratic elution (small molecules < 1000 g/mol) or a suitable gradient for the separation of peptides or other larger molecules.

Gradient elution can also be chosen for separation of compounds that vary greatly in their polarity. As a rule of thumb, if peaks can be detected during more than 25% of the scouting gradient, gradient elution will likely be the best choice for that separation problem. If peaks appear for less than 25% of the scouting gradient, isocratic elution should be preferred, as the selectivity will always be superior under isocratic conditions.

If the sample consists of two distinct groups of compounds, step elution can lead to a good separation results. In this case one starts with isocratic mobile phase elution that renders satisfying separation results for the less retained group of compounds, followed by increasing the elution strength of the mobile phase in one step to such a degree that will separate the more retained group of compounds in an appropriate way. Step gradients can also be used for washing the column after every injection. In this case the elution strength is increased drastically after the last peak has eluted. Strongly retained impurities can then be removed from the column.

21. Which are common buffers used in HPLC?

pH buffers

Salt pKABuffer

pH rangeequilibrium

UV cut-off

Ammonium acetate4.76 9.2

3.8-5.8 8.2-10.2

HAc ↔ Ac− NH4

+ ↔ NH3205

Ammonium formate3.8 9.2

2.8-4.8 8.2-10.2

HCOOH ↔ HCOO− NH4

+ ↔ NH3200

Potassium phosphate2.1 7.2

1.1-3.1 6.2-8.2

H3PO4 ↔ H2PO4−

H2PO4− ↔ HPO4

2− 190

Triethylammonium acetate

4.76 11.01

3.8-5.8 10.-12.0

HAc ↔ Ac− Et3NH + ↔ Et3N

235

Additives

Salt pKABuffer

pH rangeequilibrium UV

cut offAcetic acid 4.76 - HAc ↔ Ac− 205Formic acid 3.8 - HCOOH ↔ HCOO− 200Trifluroacetic acid 0.3 - F3CCOOH ↔ F3CCOO− 210

Trietylamine11.01

- Et3NH+ ↔ Et3N 235

Diethylamine 10.5 - Et2NH2+ ↔ Et2HN 235

Ammonium hydroxide

9.2 - NH4+ ↔ NH3 190

22. Which chiral column should be used?

It is nearly impossible to foresee which chiral stationary phase will provide the highest selectivity for a certain racemate. Some common racemates that can be separated by our chiral column can be found in the application guide. If the structure of your compound is similar to the compounds shown in the application guide, then there is a good chance that the same column / mobile phase will also provide enantiomeric separation for your compound.

Kromasil AmyCoat and CelluCoat are stationary phases that are very general, thus provide separation for most racemates. These two columns can be used under normal phase, polar mode and reversed phase conditions. For reversed phase applications, specific RP columns should be used. Kromasil AmyCoat and CelluCoat are based on modified polysaccharides that are adsorbed onto a silica matrix. Attention has to be given to the choice of mobile phase solvents in order to avoid desorption of the polysaccharide polymer.

Kromasil TBB and DMB are less wide in their application, but on the other hand, they can be used with any solvent as mobile phase. Kromasil TBB shows generally good separations for acidic racemates. Kromasil DMB and TBB can be used under normal phase conditions. Best results are often obtained when an aprotic polar modifier is used, e.g. ethers or esters incombination with heptane. For basic racemates, acidic and basic additives often lead to significant improvements of the peak shape. The acidic additive (e.g. formic acid or acetic acid) should be used in excess, e.g. 0.1% acid and 0.05% amine (DEA, or TEA).

For detailed information concerning our chiral columns, please consult our chiral brochures.

23. Which detector flow cell should I use for my column?

In order to get the best possible sensitivity, it is important to work with the correct flow cell / column combination. Standard HPLC systems are optimized for 4.0 to 4.6 mm ID columns. If smaller ID columns are used, the standard detector flow cell will likely contribute to the extra column volume to such an extent that peak efficiency is compromised. The table below indicates combinations of detector flow cells and column dimensions:

Column ID [mm]Typical flow rate

[mL/min]Flow cell volume [μL]

4.6 1.0 13-154.0 0.75 13-153.0 0.5 5-92.1 0.2 5-91.0 0.05 1-2

24. Which Kromasil product corresponds to the USP column nomenclature?

USPDescription Kromasil products

L1Octadecyl silane chemically bonded to porous silica

Kromasil 100 Å C18 Kromasil 300 Å C18 Kromasil Eternity C18

L3 Porous silica microparticlesKromasil 60 Å SIL Kromasil 100 Å SIL Kromasil 300 Å SIL

L7 Octyl silane chemically bonded to porous silicaKromasil 100 Å C8 Kromasil 300 Å C8

L8An essencially monomolecular layer of aminopropylsilane chemically bonded to totally porous silica

Kromasil 100 Å NH2

L10Nitrile groups chemically bonded to porous silica microparticles

Kromasil 60 Å CN

L11Phenyl groups chemically bonded to porous silica particles

Kromasil 100 Å Phenyl Kromasil Eternity PhenylHexyl

L13Trimethylsilane chemically bonded to porous silica microparticles

Kromasil 100 Å C1

L26Butyl silane chemically bonded to totally porous silica particles

Kromasil 100 Å C4 Kromasil 300 Å C4

25. Which mobile phases work with my detection wavelength?

Certain buffers or organic solvents interfere with UV detection. Only mobile phases with a UV cut-off below the detection wavelength will not compromise the signal sensitivity.

The tables below show the UV cut-off of the most commonly used buffers, solvents and additives in HPLC.

pH value Buffer UV cut-off [nm]2.0 – 3.0 Phosphate2103.5 – 5.5 Acetate 2404.0 – 6.0 Citrate 2506.0 – 8.5 Phosphate2107.0 – 9.0 TRIS 2258.0 – 10.5Borate 210

Solvent UV cut-off [nm]Acetone 330Acetonitrile 190Dichloromethane 233Dimethoxy sulfoxide 268

Solvent UV cut-off [nm]Dioxan 215Ethanol 210Ethylacetate 256Hexane 195Heptane 200Methanol 205Methyl-ethyl ketone 329Methyl t-butyl ether 210N,N-Dimethylformamide268Propan-2-ol (IPA) 205Tetrahydrofuran 210Toluene 284

AdditiveUV cut-off

[nm]Acetic acid 230Diethylamine 210Formic acid 210Triethylamine 235Trifluoroacetic acid210

26. Why does the chromatogram show peak tailing?

Peak tailing can occur due to numerous reasons. The problem can be identified according to the following scheme:

Mass overload: when injecting less sample amount (mass) either the peak becomes more symmetrical or resolves into two separate peaks.

Corrective action: use a more dilute injection sample.

Secondary interactions: when injecting a neutral compound (acetophenone, toluene) the peak becomes symmetrical

Corrective action: adjust the mobile phase pH in order to render neutral analytes.

For larger ID columns (>10 mm) radial temperature gradient can also cause peak tailing. In order to avoid such, it is recommended to use a column oven. The column should be 1-2°C warmer than the mobile phase in order to compensate for the friction heat generated in the core of the column.

If neither of the above applies, the tailing can also be caused by irregularities in the column packing process or by a partially clogged inlet frit leading to an inhomogeneous flow profile.

27. Why does the chromatogram show poor efficiency (plate count)?

1. Injection amount / injection volume

Overloading by mass or volume as well as the wrong sample solvent can decrease the efficiency of the column significantly and therewith impair the results of the separation. The injection volume should not exceed 10% of the flow rate (e.g. 1 ml/min ? injection volume < 100 µL. Mass overloading is obtained as soon as the linear part of the adsorption isotherm is surpassed. This border is individual, but as a rule of thumb, no more than 0.01 mg sample should be loaded per ml column volume (e.g. max 22 µg on 4.6 × 250 mm column). The sample mass may however be increased if necessary for detection reasons. In the sample solvent, the amount of the strong mobile phase component (alcohol, acetonitrile in RP, or alcohol, ethyl acetate etc. in NP), should not exceed the amount in the mobile phase.

2. Extra column volume / dead volumes

Poor plate counts can often be attributed to excessive dead volume. The dead volume is defined as the sum of the volume contributed by the sample injector, including the sample loop, the tubing connecting the sample injector to the column, the tubing connecting the column to the detector flow cell, the detector flow cell, plus any volume added by fittings, connectors, and in-line filters. Tubing with 0.010" ID is commonly found in HPLC systems. If you are using narrow bore (< 3.0 mm ID) columns, or, you can enhance your system´s performance by replacing the 0.010" ID tubing with 0.007" ID tubing to reduce system dead volume. Tubing with 0.005" ID can further reduce dead volume, but at a cost of inconvenience. Tubing with IDs less than 0.007" restricts flow rate and may have problems with buffer salt precipitation. If you use a variety of brands of HPLC columns, make sure that the fittings used to connect to the column are properly matched. If your system has stainless steel tubing and fittings, select the proper fittings for each brand of column that you use and install those fittings prior to connecting the column. If your system has PEEK tubing and fittings, it is a good idea to install a new PEEK fitting on a freshly cut piece of PEEK tubing with each column change to insure a proper fit.

28. Why does the chromatogram show split peaks?

Split Peaks can be caused by:

Column contamination Partially plugged frit Column Void Injection Solvent Effects Co-eluting compounds

Column contamination or partially clogged frits are generally caused when unfiltered samples are injected or when the sample solvent is different to the mobile phase. If the latter is the case, constituents of the sample may precipitate at the column inlet upon mixing with the mobile phase or by contact with the metallic surface of the frit.

A column void is formed either by hydrodynamic stress (high flow rate, high viscosity of the mobile phase) of a poorly packed column, by mechanical impact (the column was dropped on the floor) or by chemical dissolution of the packing material, e.g. pH > 12.

When using injection solvents that have higher elution strength than the mobile phase split or broader peaks can appear. The effect is most pronounced for early eluting peaks.

Split peaks can also occur by either two compounds that are almost co-eluting. If this is the case, the peaks should separate better if a column with higher efficiency (theoretical plates) is used. This can either be achieved by smaller particle size or a longer column. Also higher temperature and/or a less viscous mobile phase may promote a better separation.

Moreover, one compound can elute in two states, e.g. if not sufficient ion-pairing reagent is present in the mobile phase or the buffer capacity is deficient.

29. Why is it difficult to get reproducible retention times in normal phase chromatography?

The reason for the variability in retention time in normal phase HPLC is the strong dependence of the retention on the content of highly polar mobile phase constituents, especially water. Even if no water is added to the mobile phase on purpose, small quantities will always be dissolved even in very apolar solvents. Furthermore, bare silica is extremely hygroscopic, thus water will be adsorbed on the surface. Hence, the water content in a normal phase system can vary significantly and is seldom controlled. Best results, e. g. reproducible retention times, are obtained when working with half-saturated mobile phases. A straight forward way to obtain a half saturated mobile phase is to divide the mobile phase in to equal parts. To one part (e.g. 500 mL), one adds 2-3 mL water and allows the mixture to stir for about 30 min. Thereafter the excess water is removed (separating funnel), and the dry part (500 mL) is added to the saturated portion.

Even if a half saturated mobile phase will reduce the time to reach equilibrium in the column to a large extent. The equilibration of a normal phase column, especially when dealing with bare silica, can still take hours. Furthermore, polar modifier stationary phases, such as cyano or diol, are generally much less prone to variations in the water content of the mobile phase and can be equilibrated faster.

Some examples of solubility data of water in organic solvents at 25°C.:

Heptane: 0.0091% (w/w) Ethyl acetate: 2.94% (w/w) Toluene: 0.0334% (w/w) MTBE: 1.50% (w/w)