General info-08

Guard columns

In HPLC, Guard columns are mostly used to extend the life of the analytical columns. Guard column is a small sized column packed with the same packing material as like analytical column. Since the frits on guard column also remains same as those on analytical column, the particles blocking the analytical column will block the guard column first. So the analytical column will be safeguarded by the use of guard columns.

(Analytical columns- Columns like IEX, RPC, SEC, HIC normally used for the purification and analysis of samples)

RPC-04

Use of Ion Pairing reagents in RPC

Ion pairing reagents are mainly used in RPC to make the analytes to interact with stationary phase (refer previous RPC posts). Ion pairing reagents will bind to the ionic molecules in the analyte via ionic interactions which make the hydrophobic groups present in the analyte to be exposed out for the affinity of binding with stationary phase. Trifluoroacetic acid is mostly used as an ion pairing reagent for the reversed phase separation of peptides (Glajch J L et al, 1987) (Shibue M et al, 2005). Here, the negatively charged trifluoroacetate interacts with the positively charged amino acid residues and makes the analyte less hydrophilic. (Geraldine Thevenon, 1989) 

IEX-03

Effect of Isoelectric point on IEX

Every protein has an Isoelectric point (pI) (refer General info-01). When a protein is at its pI the protein will not even bind to the ion-exchange resin. Below this pH the protein will have a net positive charge and will bind to a cation exchanger, and above this pH it will have a net negative charge and bind to an anion exchanger. 

IEX-02

Stationary phase in IEX

The cation exchanger has sulfomethyl, carboxymethyl groups and the anion exchanger has Quarternary ammonium, Diethylaminoethyl (DEAE) groups attached to the stationary phase resin. 

IEX-01

Ion Exchange Chromatography (IEX) 

Ion exchange chromatography (IEX) separates proteins based on their net surface charge, through electrostatic interactions that occur between proteins and a charged stationary phase.

Types of IEX 

Two types of IEX exist:

1) Anion exchange (Negatively charged proteins bind to positively charged stationary phase)

2) Cation exchange (Positively charged proteins bind to negatively charged stationary phase)

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General info-07

Know about Ghost peaks 

One of the major problems in chromatography is the occurrence of ghost peaks in chromatogram. These peaks are mainly due to the contamination of column from previous run samples. So to avoid those ghost peaks, the column should be washed or flushed  properly with strong solvents at the end of every runs.

RPC-03

Endcapping

During manufacture of RP columns, some of the silanol groups remain on the surface of the silica after immobilization of non polar functional groups. This is because of the steric hindrance from C8 or C18 chains. Basically the stationary phase of  RPC should be non polar but the silanol groups are polar. This leads to the chance of interaction of unwanted analytes with stationary phase resulting in poor peak resolution and peak tailing. End capping is used to mask this interaction by replacing silanol groups with trimethylsilyl group.The reagents like trimethylchlorosilane and dichlorodimethylsilane are commonly used for end capping the reverse phase columns.