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Assignment HPLC

Retention time, the time required for a compound to pass through an HPLC column, can shift between trials and days due to several factors. Temperature changes of even 1°C can cause a 1-2% shift in retention time. Increased back pressure in the column from contamination can also shift retention time. pH must be carefully controlled, as even a 0.1 change can result in a 10% retention time change for ionizable compounds. Column aging, contamination, improper mobile phase composition, or flow rate changes can additionally impact retention time consistency over time. Careful control and monitoring of these method parameters can help reduce variability in retention times.

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Rahat
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55 views

Assignment HPLC

Retention time, the time required for a compound to pass through an HPLC column, can shift between trials and days due to several factors. Temperature changes of even 1°C can cause a 1-2% shift in retention time. Increased back pressure in the column from contamination can also shift retention time. pH must be carefully controlled, as even a 0.1 change can result in a 10% retention time change for ionizable compounds. Column aging, contamination, improper mobile phase composition, or flow rate changes can additionally impact retention time consistency over time. Careful control and monitoring of these method parameters can help reduce variability in retention times.

Uploaded by

Rahat
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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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 2. a. Increase run time or gradient slope
injection b. Increase flow rate
3. Vacancy or ghost peaks 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 2. Change solvent; whenever possible, inject samples in
phase mobile phase
3. Contamination on guard or analytical column 3. Remove guard column (if present) and attempt analysis.
inlet. Replace guard column if necessary. If analytical column is
obstructed, reverse and flush. If problem persists, column
4. Partially blocked frit. may be clogged with strongly retained contaminants. Use
appropriate restoration procedure (Table 2). If problem still
5. Small (uneven) void at column inlet. persists, inlet frit is probably (partially) plugged. Change
frit or replace column.
6. Sample solvent incompatible with mobile
phase. 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.

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