Tuesday, February 28, 2012
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.
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.
HILIC COLUMNS AND THEORY DISCUSSED LATER ON...............
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.
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.
MORE WILL BE DISCUSSED LATER ON........................
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.
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.
MORE WILL BE DISCUSSED LATER ON.................
4. 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.
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.
5. 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.
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.
6. 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.
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.
7. 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.
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.
8. 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.
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.
LOTS OF POINTS NEEDS TO BE DISCUSSED IN VERY DETAILS................THOSE WILL BE DISCUSSED AS SINGLE TOPIC LATER...............KEEP FOLLOWING..........
YOURS CHOROMATOGRAPHICALLY,
KAUSHIK ZALA
