General Questions - Tips & Techniques:
Ask Dr. Maria
- What is “Aqueous-Normal Phase” Chromatography?
- What is an Octanol/Water Partition Coefficient?
- What is a Ballistic Gradient?
- What can cause excessive back pressure?
- When should I use an Amino Column?
- What is Dwell Volume?
- What are the advantages of using a 3.0mm ID HPLC column for LCMS?
- What determines the retention or hydrophobicity of a molecule in HPLC?
- What life time should I expect from a Cyano Column?
- Why does the metal content of a silica particle contribute to the peak shape of my chromatogram?
- Should I filter my HPLC Solvents and Buffers before use?
- Why do some methods use low pH buffers to separate acidic compounds?
- I am confused when it comes time to use Phenyl, Amino or Cyano Columns. What are the main differences?
- What is the first step that you would recommend in developing a new HPLC method and where should I start?
- What performance standards should I set for a routine method? How do I go about developing this?
- I heard that rapid changes in column back pressure can damage the column. Is this true?
- What special care should I take to protect my columns when I am equilibrating them?
- What are the typical flow rates recommended by HPLC column companies to start with, for various column ID’s?
- What would you recommend to reduce time to develop an HPLC method?
- What is a "system peak"?
- 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?
- What is the maximum changes I can make to a method to increase the chances of meeting system suitability in HPLC?
- Can I use trifluoroacetic acid (TFA) to improve the chromatographic performance in LC-MS analysis?
- 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?
- 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?
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
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
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
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.
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
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
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.
Recommended Flow Rate
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
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.)
|Concentration of buffer salts
|pH of Mobile Phase
|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.
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.