Dear Chaitanya,

i believe you are using UV detector.

1. the negative peak is due to solvent UV cut off of your mobile phase. if the solvent UV cut off of your mobile phase is higher in your interested wavelength, the analyte may have less aborbance than the mobile phase in your selected wavelength , the peak will go negative side as we are zeroing the baselinbe with mobile phase. if this is the case change wavelength / mobile phase UV cut off

2.or, the selection of polarity in the D2 lamp may be in -ve mode. if so please change into + mode.

3.Use of different buffers in HPLC:

use buffer or modifier if the analytes are ionisable because ionisable compounds will show as two peaks or split peak as it is existing in two form (HA and A-); so if you buffered the mobile phase into basic or acidic the analyte will go to single form. maximum use acidic pH (2-4) as the mostly column will hydrolyse more in basic condition

HA------------> H+ + A-

0.1% TFA, H3PO3, H2SO4, 1-2 % Acetic acid are the simple modifiers to restrict the pH into acidic side to improve the peak shape) and well known phasphate buffer (10 mmol – 100 mmol phasphate buffer), optimum is 30-50 mmol

or

Please read the below article

http://www.laserchrom.co.uk/LaserchromHPLC-pHBufferGuide.htm

Regards

Selvan

Dear Chaitanya,

i believe you are using UV detector.

1. the negative peak is due to solvent UV cut off of your mobile phase. if the solvent UV cut off of your mobile phase is higher in your interested wavelength, the analyte may have less aborbance than the mobile phase in your selected wavelength , the peak will go negative side as we are zeroing the baselinbe with mobile phase. if this is the case change wavelength / mobile phase UV cut off

2.or, the selection of polarity in the D2 lamp may be in -ve mode. if so please change into + mode.

3.Use of different buffers in HPLC:

use buffer or modifier if the analytes are ionisable because ionisable compounds will show as two peaks or split peak as it is existing in two form (HA and A-); so if you buffered the mobile phase into basic or acidic the analyte will go to single form. maximum use acidic pH (2-4) as the mostly column will hydrolyse more in basic condition

HA------------> H+ + A-

0.1% TFA, H3PO3, H2SO4, 1-2 % Acetic acid are the simple modifiers to restrict the pH into acidic side to improve the peak shape) and well known phasphate buffer (10 mmol – 100 mmol phasphate buffer), optimum is 30-50 mmol

or

Please read the below article

http://www.laserchrom.co.uk/LaserchromHPLC-pHBufferGuide.htm

Regards

Selvan

Thanks for ur intrest in replying me

on what basis we select buffer and ion pairing agent in hplc methods