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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You may also like this
How to select a column for HPLC method development
Principle and Procedure of HPLC
Why is Gradient Elution used in HPLC?
Isocratic and gradient elution in HPLC
Difference between isocratic and gradient HPLC system
Advantages and Disadvantages of Gradient Elution in HPLC
Advantages and disadvantages of isocratic elution in HPLC
Advantages of Gradient Elution over Isocratic Elution
Difference between isocratic and gradient elution
Advantages of isocratic elution
How many types of pumps are there in HPLC
Sample Injection System in HPLC
Rheodyne injector in HPLC
Injection volume in HPLC
Effects of Injection volume in HPLC
HPLC column loading capacity
How to Determine Loading Capacity
HPLC Injector and Types of HPLC Injector
How to increase peak response in HPLC
what are the causes of peak tailing and fronting
Thanks bro
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