Question

Describe for the following HPLC procedures: (1) What is meant by? (2) What is the purpose of? (3) How is it done?

(a) Degassing the mobile phase

(b) Filtering the mobile phase

(c) Modifying the mobile phase

(d) Endcapping the stationary phase

(e) Pulse-dampening the mobile phase

(f) Introducing a Guard column

(g) Introducing a Suppressor column

Answer 1

a)

1) degassing is the removal of atmospheric gases dissolved in the solvent. 2) degassing is done so that when the gas encounters nucleation site inside the instrument it may not have bubble formation. Bubbles formation interfere with the flow of solvent in the instrument 3) degassing is done by purging the solvent with helium gas which removes substantial amount of dissolved gases.

b)

1) the solvent is filtered through a fine 0.22 micron filter to remove any visible translucence or dust particles that are not visible to the naked eyes. 2) this is done to decrease the chances of instrument wear and tear and also to increase the life of the instrument filters 3) this is separately by a filteration unit in which a disk filter of appropriate pore size is fitted and the solvent is run through it using suction pump.

c)

1) this means changing the amount of organic modifier used in the eluent solution so that its hydrophobicity/ hydrophilicity can be altered. 2) this is done to vary the retention rate of the sample in the column 3) this is a hit and trial technique and is usually done at research level. simply involves changing the percentage composition of the eluent and running hplc each time and standardising the protocol at which the best results are obtained.

d)

1) endcapping is the replacement of accessible silanol groups in a bonded stationary phase by TMS or trimethylsilyl groups. 2) purpose is to reduce the tailing of the compound so that we may get almost pure separation of the samples 3) endcappiing is done mostly by using a 1:1 molar ratio of trimethylchlorosilane and hexamethyldisilazane

e)

1) pulse dampening means that the solvent incoming pulse is stabilised by the instrument or dampener. 2) purpose is to prevent the building of uneven pressure by the solvent pulse, it serves to equalise the pressure created by the pulse 3) pulse dampener is a special instrument fitted inside the hplc which regulates the pulse of mobile phase.

f)

1) guard column are a type of filters that are fitted in the hplc instrument 2) their purpose is to protect the analytical column from the chemical impurities in the sample 3) they are set between the injector and an analytical column

g)

1) these are also a kind of filters, 2) that chemically reduce the eluent background conductance by removing carbonates and bicarbonates from the mobile phase. it also increases the electrical conductance of the analyte ions. 3) these columns are also attached to the hplc instrument whose functioning can be controlled using the instrument monitor.