Sunday, December 29, 2019

How to select a column for HPLC method development

In high-performance liquid chromatography, to separate the components is our object and this occurs in the column (Stationary Phase), hence the column is the heart of the HPLC system. Changing the HPLC columns during method development will have the most impact on the resolution of the analytes. Typically, current reverse phase chromatography columns are made by packing with globular silica gel beads that are coated with the hydrophobic stationary phase. Typically the nature of the stationary phase has the most influence on the elution, capacity factor, selectivity, and efficiency. There are various types of matrices for stationary phase support, including polymers, silica, and alumina.

Silica is the most regular matrix for HPLC columns. Silica is chemically stable for low pH systems and most organic solvents. The drawback of silica solid support is that it will dissolve above pH 7. Nowadays HPLC columns are developed for use in high pH range. The particle size, nature, and shape of silica effect the separation of analytes. The use of small particle size of silica increases the separation efficiency or increases the number of theoretical plates. But, the use of small particles increases the backpressure of the system and the column becomes more easily plugged. The mobile phase in RP-HPLC is polar and the stationary phase is non-polar, whereby polar molecules are usually eluted earlier than non-polar molecules.

To form a stationary phase for RP-HPLC on silica supports, to introduce a non-polar surface free silanols are reacted with a chlorosilane with hydrophobic functionality. Because of static barriers, only about 1/3 of the silanols are derivatized. The remaining silanols may interact with the molecules, resulting in peak tailing. Typically, after column derivatization with the preferred stationary phase, chlorotrimethylsilane reacts with column STEM to eliminate the remaining free silanols and improves the efficiency of the column. C18 (octadecyl), C4 (butyl), Cs (octyl), phenyl (phenyl propyl) and nitrile (cyanopropyl) column are commonly used stationary phases. In common, higher carbon loads higher phase loadings, and longer alkyl chains gives better retention of non-polar components.

Here are listed some common bonded phases for HPLC columns.
SI, C1, C2, C3, C4, C5, C6, C8, C18, CN, NH2, NO2, OH, PHENYL, SCX, SAX, WCX, WAX.


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Difference between dissolution and chemical reaction

Dissolution is the process while a solute is completely dissolved in a solvent. The chemical reaction is any chemical change in a substance that involves a change of one or more matter with completely different properties is called a chemical reaction.

The main difference between dissolution and chemical reaction is that the dissolution is used to determine the rate of oral dosage form or solute in a medium or solution. While in chemical reactions the reactants are modified to make the chemical with completely different properties of the reactant.
 

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Difference between NMR and IR spectroscopy

Spectroscopy is the study of the interaction between electromagnetic radiation and matter. Depending on the energy of light employed, the sample contains different types of excitation and it serves as a means of classifying different spectroscopy methods.

IR gives information about the functional groups in a sample component in the range of 400-4000 cm−1. NMR spectroscopy used to study the absorption of radiofrequency radiation by nuclei in a magnetic field. This method uses radio waves.

FT-IR and NMR spectroscopy can give chemical information about sample molecules. The main difference between NMR and an IR spectroscopy is that the NMR spectroscopy is used to identify organic structure while FT-IR spectroscopy used to determine the presence and absence of functional groups in the sample.


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Applications of LC-MS

The LC-MS technique uses an HPLC, in which the individual analytes in the sample mixture are first separated after ionization and the ions are separated based on their mass/charge ratio. The separated ions are after that directed through an electron multiplier tube detector, which recognizes each ion and determines its quantity. Mass spectrometry (MS) has played an essential role in many stages of the drug discovery process to identify and characterize lead molecules.
LCMS is useful in several industries for example biopharmaceutical, pharmaceuticals, food, forensic, and environmental sectors. Some applications of liquid chromatography-Mass spectrometry (LC-MS) are mentioned below.
Pharmaceuticals applications of LC-MS:
LC-MS is broadly used in the determination of pharmaceuticals and particularly in the separation of alternatively active drugs. Separation, detection, and purification of drug metabolites is one more major application of LC-MS. Separation and characterization of molecules in a sample mixture of natural products for example alkaloids, complex lipids, and fatty acids using LC-MS has been performed.
Quality Control:
Liquid chromatography-Mass spectrometry is a suitable technique for detection testing using a reference standard and for determining the purity of a drug substance or product.
Pharmacokinetic use of LC-MS:
Bioanalysis is commonly performed by an LC-MS / MS instrument since they have much higher sensitivity and identification as compared to UV detectors which are commonly used in high-performance liquid chromatography.
Environmental Applications of LC-MS:
This technique is employed in the analysis of various samples such as soil, sludge, air and drinking water or wastewater, etc.
Samples can belong to numerous different chemical classes, ranging from non-polar hydrocarbons to ionic organonomic classes.
Biomedical Applications of LC-MS:
The LC-MS method is functional for detecting steroids in body fluids. The high sensitivity detector using this method steroid sulfate can detect.


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LC-MS compatible buffers

Solvents or mobile phase is usually selected based on solubility and compatibility of a compound with various ionization techniques used in LC-MS. The volatility and ability of a solvent to donate a proton are significant in ESI and further atmospheric ionization methods. The special considerations should be taken when selecting a buffer for liquid chromatography-mass spectrometry applications. The volatile buffers are needed to avoid fouling of the API interface an increase in buffer concentration in both positive and negative ESI may lead to a decrease in the signal of the molecule. However, this effect is dependent on some molecules that only show a small loss of response.
Below listed are the compatible buffers (volatile) in LC-MS.
  • Trifluoroacetic acid (TFA) (pKa-0.2)
  • Formic acid (pKa-3.8)
  • Ammonium Formate (pKa-3.8)
  • Acetic acid (pKa-4.8)
  • Ammonium acetate (pKa-4.8)
  • Ammonium Formate (pKa-9.2)
  • Ammonium acetate (pKa-9.2)
  • Ammonia (NH4OH) (pKa-9.8)
The buffer is more effective when used in ± 1 pH unit of its pKa, however adequate buffering from the pKa can provide the ± 2 pH unit. Always use the highest quality and properly filtered buffer solutions. A buffer concentration of up to 25 mM will be adequate for LC-MS analysis. The accurate concentration of buffer is dependent on the analysis goals, lower concentrations of the buffer will typically increase column lifetime. It is recommended to avoid the use of phosphate buffer in the analysis of LC-MS, as it has low volatility.


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Mobile phase selection in LC-MS

The use a volatile mobile phase is a good starting point. As well as essential mobile phases such as methanol, water, acetonitrile, and acetic acid are also generally used to regulate pH levels. For buffer solutions, ammonium acetate and ammonium formate are commonly used since they are volatile salts. Besides, to make easy the separation of polar analytes by reversed-phase liquid chromatography, the volatile ion-pair reagents can be added to the mobile phase. These reagents have a polar ionic group and a long hydrophobic tail, which will attach to the stationary phase. In the presence of ion-pair reagents, the polar molecules interact with the charged ionic groups of the ion-pair reagent and are separated in the reversed-phase HPLC column. With a focus on ionization efficiency, solvents are necessary for producing reaction ions for APCI, and polar solvents are required for ESI because they are essential to dissolve polar molecules.
The most generally used solvents consist of LC-MS grade acetonitrile and water, including ion pairing agents such as formic acid, trifluoroacetic acid. Below are some examples of the mobile phases which are used in the analysis of LC-MS.
  • Trifluoroacetic Acid (TFA)
  • Formic Acid (FA)
  • 0.1% TFA in Acetonitrile, 
  • 0.1%TFA in Water, 
  • 0.1% FA in Acetonitrile
  • 0.1% FA in Water
Try to keep away from using phosphate buffers in liquid chromatography-mass spectrometry as there is the accumulation of in-volatile species, so they block sample orifice.
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