What is the most effective way to clean HPLC columns after or before use?

Many commonly used cleaning methods feature a strong wash solvent, run isocratically in order to elute strongly retained compounds or residual solvents from the column. After some time of washing in this manner, the stationary phase is essentially in equilibrium with the wash solvent, so it may still take a long time to remove contaminants from […]

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What is trifluoroacetic acid (TFA) and when should I use it?

TFA is an abbreviation used for trifluoroacetic acid. This acid is commonly used in HPLC methods because it has some ion-pairing characteristics and is therefore used where peak tailing due to causes such as silanophilic effects is an issue. It is generally considered to be not compatible with LC-MS except when used in very low […]

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What will the pressure be with a mobile phase of 90% methanol/ 10% DI water with a 4.6 x 250mm Cogent TYPE-C™ HPLC column?

There are a number of points to address in answering this question: • Factors that affect pressure. In addition to the mobile phase composition, a number of other parameters will determine the pressure. These include flow rate, temperature, and instrument tubing. Therefore it is difficult to say what the pressure will be in general cases. Using a 4.6 x […]

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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. […]

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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. […]

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When to Use PEEK or Stainless Steel for HPLC Column Hardware – Tips

Data in certain types of analyses may be adversely affected by use of stainless steel HPLC columns. In these cases, you may want to consider a column made of PEEK hardware instead of traditional stainless steel. Mainly used in Ion Chromatography and for some bioactive compounds, PEEK columns may lack column to column precision and […]

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Where a Static Mixer for HPLC Instrument Should be Placed.

Static Mixers are designed to reduce Detector Noise associated with heterogeneity of multiple mixed Solvent sources by mixing more thoroughly than can be accomplished with a proportioning valve in the HPLC Instrument. The best place for the Static Mixer is after the solvents have been mixed by the Instrument but before the Sample has been […]

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Which Filter Membrane is best for Aqueous Buffers? FAQ

The “Best Membrane” is a difficult Question to answer as it depends on your Sample that you are Filtering and the Solvent is it dissolved in. For most HPLC and Dissolution testing, Hydrophilic Nylon Membranes are generally used because they are durable, compatible with most Aqueous Solvents including Water and have the lowest Extractables with […]

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Which membrane is best for aqueous buffers when using Syringe Filters?

The best membrane is a difficult question to answer as it depends on your sample that you are filtering and the solvent is it dissolved in. For most HPLC and Dissolution testing, hydrophilic Nylon membranes are generally used because they are durable, compatible with most aqueous solvents including water and have the lowest extractables with […]

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Why am I Seeing Multiple Peaks in HPLC for Fructose?

I am running an HPLC method for Fructose but I see two or more peaks. What could be the cause? Suggestion: The peaks are likely due to the α and ß anomers as well as pyranose and furanose forms. In the open chain form, either a five-member or six-member ring can form. From there, the […]

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Why ANP HPLC Methods are Better to use than HILIC.

Why ANP is better than HILIC for Polar Compound Methods Compared to ANP, HILIC will require more time to Equilibrate, takes more salt to retain some compounds, lacks Retention Time (RT) Precision and Lifetime of the Columns can be very short. With Cogent TYPE-C Columns using ANP, Equilibration is extremely fast, Retention Time (RT) Precision […]

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Why Aqueous Normal Phase is better than HILIC for most polar compounds in HPLC.

HILIC will require more time to equilibrate, more salt to retain compounds, lacks RT precision and lifetime of the columns is short. With ANP equilibration is extremely fast, RT precision is extraordinarily good and columns last up to 10x longer. Also ANP can retain some non-polar compounds.      These columns can be used for reverse (RP) or normal phase […]

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Why do I have Higher Pressure at a Lower Flow Rate in my Results?

All other factors being equal, the Pressure should not increase when the Flow Rate is lower. Therefore there must be something else causing the increased Pressure. Without more detail on the situation, it is difficult to determine with certainty. Some Possible Explanations are: • Blockages in the frits. If this is the case, use a disposable […]

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Why is isopropanol rarely used as a solvent in HPLC methods?

There are several reasons. First, IPA is more viscous than most solvents and therefore produces a higher backpressure. As such, it limits the applicability of the method to lower flow rates and is therefore less useful. Second, there are generally few selectivity advantages to IPA. In a selectivity triangle with acetonitrile, methanol, and tetrahydrofuran at […]

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Why is isopropanol rarely used as a solvent in HPLC or LCMS methods?

There are several reasons.  First, IPA is more viscous than most solvents and therefore produces a higher backpressure. As such, it limits the applicability of the method to lower flow rates and is therefore less useful. Second, there are generally few selectivity advantages to IPA. In a selectivity triangle with acetonitrile, methanol, and Tetrahydrofuran at the […]

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Why use Ammonium Acetate in ANP and LCMS

Ammonium acetate can be a very useful additive for HPLC methods. With ammonium acetate, a near-neutral pH is obtained with this additive, so it is a popular choice for keeping most organic acids ionized, which is generally preferred for ANP methods. In addition, it is LC-MS compatible, in contrast to a near neutral pH phosphate […]

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Why use TEA in ANP and HPLC methods

TEA is an organic base and hence is sometimes used in some HPLC methods to adjust the mobile phase pH. Our research has indicated that TEA may be difficult to remove from columns once introduced therefore if TEA is to be used with a column, the column should be dedicated to methods with TEA. The […]

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Why you should use temperature control with the UDC-Cholesterol column

Temperature can have an effect on chromatography in general, but it becomes even more important when using the UDC-Cholesterol column. The UDC ligand is a liquid crystal, so some larger molecules interact with it in a different way than other simple molecules. The interaction is temperature dependent. Hence, to ensure robust results, please conduct your […]

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Wikipedia Reference for use of Pipets with Organic Solvents.

From Wikipedia  Piston-driven Air Displacement Pipettes These are the most accurate and precise pipettes. They are more commonly used in biology, though they are commonly used by chemists as well. The plastic pipette tips are designed for aqueous solutions, and are not recommended for use with organic solvents which may dissolve the plastic. Link to […]

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