What are the main causes of retention

time instability in reversed-phase

analysis by HPLC?
In analysis of a weak acid with pKa of 4.6, using mobile phase of phosphate buffer pH 7.7 and methanol (63:37, v/v)
and hypersil C18 column, the retention time of this analyte is not stable (it is decreasing with time), although different
types and composition of buffer and different pH were tested, but the problem was the instability of the retention time
(decrease in retention time).

Topics

Retention Times Change "For No

Reason"
Changing retention time can be a problem in HPLC, not least because we identify a peak by
its retention time. So if the retention times change, incorrect peak assignment is frequently
the result. Heres some ideas for the possible cause:

Flow Rate: First, a simple point worth noting. Under a given set of HPLC conditions
(column, temperature, eluent etc) sample components elute in a fixed volume not a fixed
time. If we can assume a constant flow rate, then this equates to a retention time. So if the
retention times of all the peaks change, the first place to look is the flow rate.

To be sure of the flow rate, measure the solvent flow to waste using a small measuring
cylinder (eg 5ml). Also watch the system back pressure. A given flow of the solvent through
the column at a given temperature generates a constant backpressure, therefore if the
backpressure changes so does the flow. Another check of the flow rate is the void volume
peak. If its position has not changed the flow rate is OK. If it has changed, the flow rate has
definitely changed. Low flow rate = longer retention times. Suspect a leak or an air bubble
first.

%B: The concentration of organic modifier in the eluent (%B) has a marked effect on the
retention time. So if the retention times change, check the %B (eg for an eluent of
water:methanol 50:50, %B = methanol concentration ie 50%). This could change because
of the method of mixing (see January 2005 Technical Tip or call give us a call on 01634 294
001) or because the ratio has changed since the eluent was made up. Things to avoid are
old eluents, bottles stood on window sills in the sun and vacuum degassing of premixed
eluents. If in doubt about the eluent, make up fresh eluent, measuring each solvent
separately before mixing.

pH: Changing the pH of a buffer solution may affect retention times. Checking this is part of
method validation, but in some cases retention times vary with pH. Bear in mind that the pH
range for most columns is pH 2-7.

Buffer Concentration (including acid concentration for ion suppression): Elution

times are almost always affected by buffer concentration. Sometimes in the case of weak
buffers, the buffer concentration is simply too low to hold the pH, but avoid high (>0.1M)
buffer concentrations wherever possible.

Stationary Phase Degradation: If the pH drops below 2 the bonded phase can be
stripped off the stationary phase. This applies to most columns using the Si-O-Si siloxane
bond to covalently attach the bonded phase. The bond is hydrolised below pH 2, turning
the column, gradually, to a silica column. In this situation you will see a gradual but
irreversible change in retention times.

Air Bubbles: If there is air in the piston chamber or the check valve of the pump the flow
rate is reduced, often in an unreproducible way. This leads to longer and variable retention
times. If this is the situation check for fluctuating back pressure (to confirm the presence of
air bubbles) and purge the system.

Leaks: If a leak develops part of the flow is diverted out of the system and the flow rate
through the column is reduced. Hence the retention times become longer. Leaks generally
occur around piston seals, tubing connectors, injection valves or from PEEK tubing if it is
kinked.

Check Valve Problems: Check valves consist of a ruby ball that allows liquid to flow
through a tube in only one direction. When the flow is in the opposite direction the ball is
pushed back into its seat where it makes a perfect seal and stops the flow. In order for this
to happen the ruby ball must be clean. Over time a surface coating can build up on the ruby
ball which prevents a good seal leading to reduced and variable flow rates. The check
valve can either be cleaned or replaced.

Temperature: Most separations are temperature dependent. Some are simply faster and
more reproducible at elevated temperatures, but for some the peak resolution is
temperature specific. So if the retention times change, check that the column heater is set
to the correct temperature.
Problems Storing HPLC Water?

The use of water in HPLC mobile phases can be a source of many problems. In this blog post we look
at the effects of storage and how to prevent storage related problems.

When you make up fresh mobile phase you probably assign an expiry date based on the fact that you
know the solvent mixture will be subject to change over time but what you may not consider is the
storage of the solvents prior to mobile phase preparation.

In the case of water, this can have significant detrimental effects on your chromatography. The
primary reason is that water is a great solvent. It can absorb contaminants via leaching from the
container in which it is stored, and it can also absorb contaminants, such as airborne bacteria, from
exposure to the atmosphere. These contaminants are likely to increase the total organic carbon
(TOC) in the water which in turn can lead to chromatographic problems such as: noisy and drifting
baselines; extra (unexpected) peaks, often referred to as 'ghost' peaks, and in extreme cases problems
with retention times and peak shapes.

The extent and impact of these effects will depend on the nature of the HPLC method that you are
using. For some methods you may not see any detrimental effects and for others it can be absolutely
critical. Contaminants are much more likely to show up, and thus create problems, if you are using
UV detection at low wavelengths, in the area of ~ 210nm, or sensitive mass spectrometry. In these
cases it would be worth investigating the effects of water storage and source (e.g., lab purification
system vs. bought in bottled water) as part of robustness in method development so that the problem
and issues are understood fully before the method is in routine use.

To prevent these problems (or reduce them as much as possible) the following is advised:

Don't store water, get it fresh before use.

 Discard the first 1-2 litres from the water purification system.
Use glass containers for water.
Don't attach plastic tubing to the delivery point of your water purification system.
Look after your water purification system, maintain it well and don't ignore warning lights!
For critical methods, investigate the effect of water storage (and source) as part of method
development.

Question:
I assess assay results for imported pharmaceutical products performed by a third party lab. The
methods we use do not always specify an "allowed" retention time range. What would a reasonable
guide be when assessing this plus or minus 10%? Basically, what I should like to know is when
should we ask the laboratory to investigate/explain a retention time shift from one assay to the next?
Put another way, when should the laboratory make system adjustments like the temperature of the
column, flow rate etc. in order for the retention time to conform? Often the method will say the
active elutes at say 10 minutes. Does this mean that 9 to 11 minutes is also OK, or are there justifiable
limits that we can impose?

Answer:
The reasons for variation of retention time from analysis to analysis includes small differences in
the following: the composition of the mobile phase (in terms of the aqueous and organic solvent
portions); the pH of the mobile phase (particularly important for ionisable molecules like acids and
bases); HPLC columns (column to column variation), and HPLC systems (particularly the system
volume). As a result of these variables it is expected that the retention time will vary and usually
small variations do not cause any problems. The potential problems associated with retention time
variation from run to run (not injection to injection in the same run, that is a more serious problem
which may indicate a problem) includes:

The change in runtime means that a peak is not fully collected by the end of the run and thus
the analysis has to be repeated.
For analysis of mixtures, such as impurity analysis, peaks may be difficult to assign if the
retention time varies.
Automatic integration events may have problems relating to identifying peaks correctly, thus
requiring extra time for reprocessing.

To answer your question, it is difficult to set an allowable retention time range without a good
understanding of the individual method. Ideally the person who develops the method should
investigate its robustness and thus gain information on the range of retention times which might be
expected due to normal variation and where the method still performs as required. Of course, it is
not an ideal world and often you will be presented with data from methods where this has not been
done, or if it has, it is not written into the method. From experience I would say that about 10 to 20%
of the time quoted in the method is a reasonable guide but be careful not to apply it too restrictively.
If you have an analysis where the retention time is on the limits, the best way to assess if there is a
problem is to look at the chromatograms for the analysis and compare to a previous satisfactory
analysis (preferably an example chromatogram should be included in the method). If there arent any
differences except for retention time, and the system suitability testing is all fine, then the results
should be satisfactory to use. If the retention time is very different to that expected and well outside
of this approximate range then I would suspect that something has been changed in the way the
method was applied and would want the analyst to investigate further.

Although pharmacopoeia sometimes include adjustments to temperature, flow rate etc. to make sure
that a peak is at a particular retention time these changes are actually deviations to the method and
unless you are following a pharmacopoeia method or have validated your method to show that these
alterations are valid then it is not a good idea to change the method parameters.

Retention Time Variability in HPLC

Im sure we have all experienced it that sinking feeling when you realize your analyte retention times have drifted
outside the software window and you have a pile of chromatograms with no quantitative results. Or you are trying to
get that system suitability result to begin your batch of analyses as you really need to get out of the door fast but your
retention times just wont settle down. Or you are trying to reproduce Bob from the R&D centers method and his
retention times (or chromatogram..!) look nothing like yours. Or you are trying to validate your method and the three
column lots you are trying give different retention behavior to the column that you just developed your method on. Or
every time you do an injection the retention time of the analyte changes just a little doesnt cause anything to fall
over, but you just dont understand why.

Yes there are a whole bunch of retention time issues that cause problems in HPLC. A lot of the underlying causes
we can do something about others we just need to be aware of the cause and put our minds to rest. The remainder
of this technical tip will outline how to overcome, or better control, all of the situations outlined above.
Overarching rules on retention time variability:

If the void (hold-up) time (t0) and analyte retention time (tR) vary together, suspect a flow rate change. In this
scenario the analyte capacity factor (k) will remain constant
If only the analyte retention time varies, with the void (hold-up) time remaining constant, then k will also
change. In this scenario suspect a change in the selectivity or retentivity of the separation system

Drifting Retention Times

This is typically due to a change in mobile phase composition, which can be caused when pre-mixed mobile phases
lose some of the organic solvent through evaporation as the run progresses. Ever noticed this seems to happen
more towards the end of a run? Well of course the organic solvent is being continually lost to the atmosphere or as
the eluent in the sealed bottle depletes there is more headspace for the more volatile component to evaporate into -
and of course a small amount of evaporation makes a bigger overall composition change in the ever diminishing
volume of liquid. This is typically why we see elution times becoming longer rather than shorter. What to do? Mix
eluents (even isocratic ones) online or at the very least ensure the reservoir you are using is capped. We may also
experience a change in the pH of the aqueous component of the eluent over time which is caused by ingress of CO 2;
lowering the eluent pH and changing the retention and perhaps even the selectivity of the separation.so again, cap
your bottle. Do not use lab film to cover eluent reservoirs especially when using MS detection (watch out for ions at
142 Da as you leach the plasticizer from the film!)

Another related note on eluents here if we de-gas pre-mixed mobile phases using vacuum, the very act of sucking
the mobile phase through the filter under vacuum can cause loss of the more volatile component which will lead to
irreproducible changes in eluent composition from batch to batch of eluent. The same is also true when degassing
pre-mixed phases using ultrasonic baths; the warming of the eluent in the sonic bath can lead to loss of the organic
modifier and, hence, change retention characteristics.

Temperature is another variable that can alter retention time, changing not only the viscosity of the eluent but the
kinetics of the retention mechanism. Ionizable compounds tend to be affected by temperature more than non-
ionogenic compounds; therefore, selectivity may also change. Most systems come with column heaters / chillers
these days, but if yours doesnt, and you get large temperature variations in the lab, this can cause retention time
variability (especially when the system is placed directly below your air-con). Even systems which do have column
heaters work in different ways some pre-warm the eluent prior to entry into the column for example and these
systems may well give different retention times to those which heat the column only.

Variable Retention Times

Equilibrating or priming a column when beginning an analysis can also show up some strange retention time shifts
and variability. Without going into too much detail, this is due to the stationary phase surface being modified by your
eluent or sample components. Primarily its the wetting of the surface (especially with more hydrophobic phases
such as C18) as the bonded phase takes on a layer of hydration - a slightly crass description but one which will do for
this short tip. Furthermore, the polar or ionized silanol (Si-OH) groups on the silica surface can irreversibly bind with
polar analyte components or buffer ions to change the overall surface polarity. What to do well you can try to inject
a 10x more concentrated sample than you normally would to try and achieve the equilibration in a shorter time (fewer
injections).

In this category we must also consider the more esoteric issue of the sample diluent. For reasons that are too
detailed to enter into here, the eluotropic strength and ionic strength of the sample diluent can sometimes have a big
effect on analyte retention time and peak shape yes thats the sample diluent, the thing you dissolve the sample in
not the HPLC eluent. You should always strive to match the aqueous / organic ratio of the eluent (at the start of the
gradient if doing gradient elution) as well as the buffer strength of the eluent. If the diluent is more highly organic than
the eluent (for solubility reasons) try to restrict your injection volume to 10 L or less.
For more information see this article:

The High Performance Liquid

Chromatography Engineering Essay
Published: 23rd March, 2015 Last Edited: 23rd March, 2015

. Liquid chromatography is a physical separation technique conducted in liquid phase. A

sample is separated into its constituent (or analytes) by distributing it between the stationary
phase and mobile phase. Mobile phase can be organic solvent like hexane and stationary
phase can be porous silica particle packed in column. High Performance Liquid
Chromatography (HPLC) is an advance form of liquid chromatography which uses small
column through which the mobile phase is pumped at high pressure.[1]

3.1.2: Theory/Principle:
3.1.3: Mobile Phase:
Most commonly used mobile phase for HPLC analysis are petroleum ether, cyclohexane,
toluene, benzene, esters, chloroform, ethyl ether, methyl ethyl ketone, alcohols, water,
organic acids. These mobile phases along with their polarity and retention time are shown in
table below:[2]

Table 1: Solvents used as mobile phase in

HPLC
Low Polarity

High polarity

Fluoroalkanes

Petroleum ether

Carbon tetrachloride

Cyclohexane

Toluene

Benzene

Esters

Chloroform

Ethyl ether

Dichloromethane

Methyl ethyl ketone

Alcohols

Water

Organic acids

Long Retention Time

Short Retention Time

Normally, the mobile phase consist of mixture of polar and non- polar solvents such as
water and ethanol.[3]

In gradient elution HPLC, the polarity of the mobile phase is continuously changed by
changing the ratio of the solvents in the mobile phase. It is most useful way to control the
retention volume or retention time of the sample component.[2]
Mobile phase must be chosen that would not interfere with the measurements by the
detector. For example, in case of ultraviolet absorption detector, the solvent cannot absorb
ultraviolet radiations. Mixture of methanol, ethanol and propanol with heptanes and
chloroform with heptanes are popular choices as HPLC mobile phases. Mixture of methanol
with water is used in reverse phase HPLC as mobile phase.[2]

Solvent Strength and Selectivity:

Solvent strength is the ability of the solvent to elute the solute from a column. The strength
of the solvent is depending on its polarity. In case of normal phase liquid chromatography,
non- polar solvent like hexane is weak solvent while polar solvent like water is strong
solvent and in reverse phase chromatography, non- polar solvent like organic solvents are
strong solvents and polar solvents like water is a weak solvent.[1]

3.1.4: Stationary Phase:

The stationary phase is a liquid film coated on a packing material consisting of 3 - 10m
porous silica particles in liquid chromatography.[4]

Some of the stationary phases used in HPLC are listed in table below: [2]

Table 2: Stationary phases used in HPLC

High polarity

Low polarity

Alumina

Magnesium oxide

Charcoal

Silica gel

Calcium oxide

Magnesium carbonate

Potassium carbonate

Sodium carbonate

Starch

cellulose
High adsorption

Least adsorption

Mostly used solid adsorbent is silica gel. The SiOH groups on silica gel make the gel weakly
acidic and attract basic compounds. Generally silica gel attracts bases in order of their
strength; i.e; strong bases are retained on silica gel columns longer than weak bases. Silica
gel is used as packing material in the form of pure particles and as a pellicle on the solid
support. A pellicle is a thin layer of coating on a surface. The pellicles used in HPLC are
chemically bonded to the surface of the support material. HPLC pellicular materials are
larger and more easily packed into the column than the micro particulate pure adsorbents
and the resulting columns are more efficient.[2]

Alumina is another solid adsorbent which is widely used in column packing material.
Alumina is basic, it retains acidic compounds. Alumina is mostly used in pellicular form.[2]

3.1.5: Instrumentation:
The basic components of HPLC are:

Solvent/ mobile supply system

Sample injection system

Columns

Detectors

Recording system

a: Solvent/Mobile phase supply system:

The basic function of solvent supply system is to deliver reproducible, precise and constant
flow of mobile phase.[8] the solvent supply system have high pressure pumps and provide
gradient elution. Solvent reservoirs are filled with solvents of different polarities, which are
miscible. The solvents used are must be pure and degassed.[7]

b: Sample injection system:

Sampling loop is widely used for the introduction of sample in liquid chromatography.[4]
sampling loops are easily changed. Loops having volume ranging from 0.5 L to 2mL are
available.[6]
c: Columns:
The high performance liquid chromatography has two types of columns.

An analytical column which is responsible for separation

Guard column which protect the analytical column from contamination

i: Analytical column:
Analytical columns used in HPLC are constructed from stainless steel material. There
length ranging from 30 mm to 300 mm and diameter between 2.1 mm to 4.6 mm. porous
silica particles are used to pack the column with the thickness of 3 - 10 m. The
efficiencies of these columns are 40,000 - 60,000 theoretical plates/m.[4]

ii: Guard column:[4]

Analytical columns have face two types of problems which decrease their life time:

Solutes binding to the stationary phase irreversibly which degrade the column efficiency by
decreasing the available stationary phase

With the sample, particulate material is injected which clog the analytical column

Before the analytical column, guard column is placed to overcome these problems.

Guard columns are less expensive; however, these are packed with the same packing
material and stationary phase as analytical column. Guard columns are replaced regularly
because they are intended to be sacrificial.

d: Detectors:
The detectors used in HPLC must have low dead volume to minimize extra column band
broadening. The detector should be small and suitable with liquid flow. The detector used in
HPLC depend on the nature of the sample.[4] [6]

The most widely used detectors in HPLC are: [4]

Spectroscopic detector

Fluorescence detector

Refractive index detector

Mass spectrometer detector

i: Spectroscopic detector:
The most widely used HPLC detectors are based on ultraviolet - visible absorption and
fluorescence.[4]

The sensitivity of ultraviolet - visible (UV) detector is about 10-8 g/ml (0.01 ppm). UV
detectors are not temperature sensitive. They are less expensive and can be used with
gradient elution.[7]

The most of the UV detectors have a simple interference filter which measures the
absorbance at few selected wavelengths. The expensive UV detectors have
monochromators that allows absorbance at particular wavelength.[6]

ii: Fluorescence detectors:

Fluorescence detectors are more sensitive as compared to UV detectors. They give
improved selectivity.[7]

iii: Refractive Index detectors:

Refractive index detectors are useful for almost all types of compounds but it has poor
detection limit. For gradient elution RI detectors are not useful. They are useful only when
mobile phase components have same refractive indexes.[4] they are less sensitive as
compared to UV detectors.[8]

iv: Mass Spectrometer detector:

Among all detectors mass spectrometric detectors are most useful detectors. It also give
structural information to identify the analyte.[4]

e: Recording system:
D:\hplc pik

3.1.6: Applications:
Purity of Samples:
Purity of samples can be determined by high performance liquid chromatography.[9]
Analysis of drugs:
HPLC is used for the analysis of drugs in biological fluids[9].

Analysis of Polyaromatic Hydrocarbons:

The concentration of polyaromatic hydrocarbons by using HPLC.[4]

Identification of unknown compounds:

Unknown compounds can be identified by comparing them with standards using their
retention times.[9]

Analysis of Biological Compounds:

HPLC is very important and useful technique for biotechnology and pharmaceutical
industries. The variety of biological compounds like DNA, carbohydrates, proteins, lipids
and amino acids are analyzed by HPLC.[8]

CHAPTER # 3
3.2: Gas Chromatography and Mass
Spectrometry: (GC-MS)
Introduction:
In gas chromatography and mass spectrometric technique (GC-MS) the components of
mixture are separated by the GC and these components are identified at molecular level by
MS.[7]

3.2.1: Principle:
In gas chromatography, when sample mixture is heated it separates into individual
components and converted into vapour states. These vapours are moved through column
along with carrier gas such as helium. The separated components emerge from the column
and enter into the ionization chamber of mass spectrometer.[7]
In mass spectrometer, molecules are ionized by using beam of highly energetic electrons.
The ionization of molecules depends upon the nature of the bonds in the molecules. The
molecular ions are broken up into many fragments. There are particular mass to charge
value for every ion. In most cases, ions have only one charge so that m/e value is the
molecular mass of the ion.[10] [11]

3.2.2: Mobile Phase:

Mobile phase or carrier gas is the important component of gas chromatography. The gas
used in GC should be inert toward stationary and mobile phase. Most commonly used
carrier gases are Helium, Argon and nitrogen. The purpose of the carrier gas is to carry the
sample through the system. Flow rate of the mobile is measured with the help of flow meter
placed at the outlet of column.[4] [8]

3.2.3: Stationary Phase:

The stationary phases used in GC should have following characteristics:[4]

It should be chemically inert

It should be thermally stable

It should be of low volatility

The most commonly used stationary phases are squalane, polydimethyl siloxane, 50%
methyl - 50 % phenyl polysiloxane, 50% cyanopropyl - 50% phenyl methyl polysiloxane,
polyethylene glycol.[4]

3.2.4: Instrumentation:
The instrument used for GC - MS consists of following basic components:

Gas chromatograph:
Inlet system

Oven

Column

Mass spectrometer
d: Ion source

e: Mass analyzer (filter)

f: Detector

g: Read out system

Injection port or Inlet system:

The function of the inlet system is to introduce the sample into the column. When the
sample is introduced it is vaporized and enters into the column along with the carrier gas.
The temperature of heated gas is maintained above the boiling point of highest boiling
component.[3] [8]

Oven:
In order to control the column temperature, column is placed inside the thermostated
over.[4]

Oven is operated on two types of analysis:[8]

Isothermal analysis.

Temperature programming.

i: Isothermal analysis:
Isothermal analysis is used when constant temperature throughout the analysis is required
i.e. there is 100C or less difference in the boiling point of low and high boiling
components.[8]

ii: Temperature programming:

When the components of a mixture have wide range of boiling point then temperature
programming occurs throughout the analysis. It gives better separation.[8]

Column:
The gas chromatographic columns are of many types such as U - shaped columns and
coiled tube columns.[7]

There are two types of columns used in GC:[7]

Packed columns

Capillary columns

i: Packed columns:
Packed columns are made of stainless steel, glass, aluminium or copper. The length of
packed column is 2 - 6 meters. Internal diameter is 2 - 4 mm.[4] The stationary phase is
packed in a column.[3] the solid support used in GC columns is diatomaceous earth. It
consist of hydrated silica gel.[4]

ii: Capillary columns:

Capillary columns are open tubular columns. Its length is about 100 meters and internal
diameter is about 0.1 - 0.5 mm. A very thin film of liquid stationary phase is coated on a
metal or glass walls of the tube. [7] Capillary column gives high separation efficiency.[4]
Capillary columns gives better resolution as compared to packed column.[3]

Ionization:
The molecules form GC column enter into ion source of MS and these sample molecules
are ionized. Ionization potential is the minimum energy required to ionize the sample
molecules.[10]

Electron Ionization

Chemical Ionization

i: Electron Ionization:
Electron ionization is the most common ionization technique used in MS. Molecules after
entering into MS bombarded with highly energetic electrons emitted from the hot filament.
After bombardment, fragments of molecules are produced. A very low pressure is required
for electron ionization process.[6] [8]

ii: Chemical Ionization:

In chemical ionization, reagent gas like methane is used. The reagent gas interacts with the
sample molecule and produce new ions. The reagent gas methane transfer the charge to
the sample molecule.[12]
Fragment ions produced in chemical ionization are much less than electron ionization
because of this reason chemical ionization is called "soft ionization technique".[13]

e: Mass Analyzer:
The ions from the ionization chamber enter into mass analyzer. The function of mass
analyzers is to resolve the different mass fragments.[6] [10]

Quadrupole mass analyzer:

Quadrupole mass analyzer is the most efficient mass analyzer used in MS. It analyzed the
analytes in less than 100 ns time scales. Du to this reason, quadrupole mass analyzer
detectors is used in GC - MS coupled technique.[14]

The quadruple mass analyzer consists of four metal electrodes. Each electrode is
connected to its opposite electrode electrically. These electrodes are 10 - 15 cm long and 5
- 6 mm in diameter. When these electrodes are polarized, ions of particular mass to charge
ratio are allowed to pass through the space between electrodes.[14]