DRUG DESIGN & DEVELOPMENT

"Why retention time of standard compound peak is shifted during different

trials and days"

Submitted to: Dr. Prof. Kashif Khan

Submitted by: Rahat Nazar

M.Phil Pharmaceutical Chemistry

The Islamia University of Bahawalpur

High Performance Liquid Chromatography (HPLC)
High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps
a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a
column with chromatographic packing material (stationary phase).

Principle:
The principle of separation in normal phase mode and reverse phase mode is Adsorption.

Retention time:
It refers to the time which is required for a compound from the moment of injection until the
moment of detection. It represents the time for which the analyte is in the mobile and stationary
phase. Retention time is substance-specific and should provide the same values under similar
conditions for a substance.

Factors like M.P, flow rate, Temperature, Column packing, Column length make it difficult to
compare retention times because these can influence retention time even if the same column is
used and small difference will remain for the same compound.

According to rule of thumb, RT is shifted during different trials by about 1% to 2% per 1°C.
Mostly the shift is caused by Increase in back-pressure in the column.

Why retention time of standard compound peak is shifted during different

trials and days?
A. Extra Peaks
Possible Cause Solution

1. Other components in sample 1. Normal

2. Late-eluting peak from previous
injection
3. Vacancy or ghost peaks
2. a. Increase run time or gradient slope
b. Increase flow rate
3. a. Check purity of mobile phase
b. Use mobile phase as injection solvent
c. Reduce injection volume
B. Split Peaks
Possible Cause Solution

1. Contamination on guard or 1. a. Remove guard column and attempt

analytical column inlet analysis
b. Replace guard if necessary
c. If analytical column is obstructed,
reverse and flush
d. If problem persists, column may be
fouled with strongly retained
contaminants
e. Use appropriate restoration procedure
f. If problem persists, inlet is probably
plugged
g. Change frit or replace column

2. Sample solvent incompatible with mobile
phase
3. Contamination on guard or analytical column 3. Remove guard column (if present) and attempt analysis.
inlet.
4. Partially blocked frit.
5. Small (uneven) void at column inlet.
6. Sample solvent incompatible with mobile
phase.
2. Change solvent; whenever possible, inject samples in
mobile phase
Replace guard column if necessary. If analytical column is
obstructed, reverse and flush. If problem persists, column
may be clogged with strongly retained contaminants. Use
appropriate restoration procedure (Table 2). If problem still
persists, inlet frit is probably (partially) plugged. Change
frit or replace column.
4. Replace frit (see above).

5. Repack top of column with pellicular particles of same

bonded phase functionality. Continue using the column in
reverse flow direction.

6. Adjust solvent. Whenever possible, inject samples in

mobile phase.

C. Distortion of Larger Peaks

Possible Cause Solution

1. Sample overload 1. Reduce sample size

D. Other Factors:

i. Temperature:

Temperature changes can also be effective in peak shifts and is an important factor.
According to rule of thumb (as mentioned above) it is about 1-2% per 1 degree Celsius.
Thermostat can be used in case if temperature is high during some analysis.

ii. Pressure:

Mostly the shift is caused by increase in back-pressure in the column. Increase in back-
pressure indicates contamination in column.

iii. pH:

If your sample is ionic or ionizable the pH of the mobile phase should be controlled very
carefully. As low as 0.1 change in pH can cause 10% change in retention time. So a well
calibrated meter should be used.
In reversed phase the retention of the acids is decreased and bases increased due to
increase in pH.

iv. Column Related problems:

Column aging, column contamination, poor column mobile phase composition for your
analyte and change in flow rate can also be some factors in changing in retention time.
These problems can be resolved by checking whether all parameters are set correctly in
Chem. Station, remaking the mobile phase and changing the column.