Column:
It is the heart of the HPLC instrument where actual separation occurs. Separation column in HPLC is usually made of heavy wall, glass lined metal or 316 grade stainless steel tubing, which could withstand high pressure and which is inert to the chemical corrosion due to mobile phase. An interior of the tubing have to be smooth along with a uniform bore diameter. Straight columns which could be operated within vertical position are preferred. A few typical tubing materials used in HPLC column are listed in Table 8.1.
Table : Column Tubing Materials and its Uses
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Material
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Use
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316 Stainless steel (SS)
Poly (ether-ether) ketone
(PEEK)
Tefzel
Titanium
Fused silica Glass
Glass
Glass-lined SS
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General utility material, good for high pressure system
Inert to most organic solvents except methylene chloride, THF, DMSO and conc. Sulphuric and nitric acids. Holds pressures up to 5000 psi (34MPa).
Good for metal-free biological systems. Inert. Common for metal-free applications.
Withstands pressures up to 5000 psi, corrosion resistant; expensive
Used for capillary LC. Limited pressure range.
Inert, withstands pressures but difficult to know when the glass is broken.
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Column fittings and connectors must be so designed that void volume is zero avoiding unswept corners. A column length ranges10 to 30 cm along with inner diameter of 2 to 5 mm giving 40,000 to 60,000 plates per inch. Therefore, shorter columns of 3 to 8 cm are also used for fast separations but in such cases, sample size will become limited. The length of the column may not only affect the resolution of a given separation -the longer the column the larger number of plates but also the speed of separation. Standard lengths vary with the manufacturer but most common values are 30, 25, 15, 12.5, 10 and 7.5 cm. It may be noted that shorter columns are described as high speed columns. The columns packed with the finer particles are more expensive than the standard 5 µ m packing.