notes on HPLC for practical pharmaceutical chemistry 802

HPLC Prelab

2008-07-05T08:50:00.001-07:00

High Performance Liquid Chromatography

A chromatographic technique used to separate a
compound between 2 phases:
solid stationary phase & liquid mobile phase
(i.e. Liquid Chromatography) done under high
pressure.
Modes of separation:
1- partition.
2- adsorption.
3- ion exchange.

Chromatography

Separation, Qualification, Quantitation: Analytical.

Separation, Purification: Preparative.

Partition chromatography is used in HPLC.

Partition coefficient is the major factor affecting the separation depending on changing of polarity of the mobile phase.

Chromatography

Gas Chromatography	LiquidChromatography
Mobile phase : Gas	Mobile phase : Liquid
Volatility of sample	Solubility of sample
M.W. < 50	M.W. > 500
Thermal Stability	Ambient temperature
Difference of boiling point	Difference of boiling point

Advantages of HPLC

separation is done at room temp or slightly elevated temperature.suitable for thermolabile substances.
Suitable for substances that can't be separate by GC [non-volatile substances]That need volatilization or derivatization to be volatile.
short time for analysis.

Elution

Elution is described by minutes Or by Capacity Factor [K]
The higher the affinity between substance [analyte] & mobile phase leads to shorter time for analysis [& vice versa].
Only compounds with different capacity factors can be separated by HPLC.

Capacity factor

Capacity factor reflects the retention time & depend on relative affinity which depends on:
1.Physico-chemical properties of the analyte.
2.Composition & flow rate of the mobile phase.
3.Surface area , particle size & column length of
stationary phase.

HPLC apparatus

The chromatographic system

Pump

transfers a specified volume of mobile phase per unit time under pressure up to 5000 psi with flow rate up to
10ml/min.
Usually one or more pump used under control of a
computer.
Pumps should be capable of delivering mobile phase at a constant reproducible flow rate. - Stability of flow rate & pressure. - Easy to use, use of variable solvents. - Pulse elimination system.

Notice for Pump operation

Degassing of solvent.
2. Do not use when solvent is absent.
3. Rinse with water after use of buffer.
4. Check Miscibility if multiple solvents.
5. Pump priming before use.
6. Pulse control.
7. Check pump pressure.

Types of elution

There are 2 types of elution :
1- Isocratic elution : fixed ratio of mobile phase
components during the analysis.
2- Gradient elution (mobile phase programming) :the components of mobile phase ratio is changed
through out the analysis for separation of components very close in polarity.

Elution

Isocratic elution for simple mixtures.
Gradient elution for complex mixture.
DO NOT used detectors that are sensitive to
change in solvent composition [Refractometer].

Isocratic & gradient elution

Mobile phase

The elution solvent (mobile phase):

HPLC grade [highly pure].

Particle free & degassed.

Water used should be of specific quality with low conductivity & low UV absorption.

Low Viscosity.

Miscibility of solvents (miscibility difference : below 15).

Solubility of the sample in the mobile phase.

Injectors

Sample dissolved in suitable solvent [with the same composition of mobile phase] giving.

particle-free solution injected into system.

Injector composed of:
Injection pore: in which needle introduced.

Fixed volume loop: which hold certain volume of the sample transferred into chromatograph.

Injector

-Manual injector (Valve injector): low accuracy.
-Automatic injector: high accuracy & speed.

Change loop volume for injection volume change

Column (stationary phase)

According to the mode of separation:
1- Normal phase:
Stationary phase (Polar)
obile phase (Non-Polar)
2-reversed phase
Stationary phase (Non-Polar)

M0bile phase (Polar)

Normal phase chromatography

Stationary phase (Polar)
Mobile phase (Non-Polar)
Non polar material elutes first.

Reversed phase chromatography

Stationary phase (Non polar)
Mobile phase (Polar)
P0lar material elutes first.

Bonded type stationary phase

Most polar column silica bonded with very polar nitrile group (CN).
Least polar column Octadecyl silane [C18]composed of a core of silica chemically bound to
an organic phase [Long chain HC].

Types of columns

Aalytical columns: of least diameter 2-5 mm & particle size = 3-10µm.
Preparative & semi preparative columns: larger diameter.
columns shouldn’t be heated more than 60C to avoid decomposition of stationary phase.

Choice of the column

Column separates mixed compounds to individual compound.
Column Selection
- Nature of the backing Material.
- Particle Size.
- Shape of the particle.
- Pore Size.

Particle Size & shape

If particle size is small → ↑↑ resolution & back pressure

Characteristics of stationary phase

• Pore size & distribution :
- Small Pore = higher efficiency = less band broadening
• Spherical or Irregular :
- Irregular = higher Surface area = higher carbon load
- Spherical = lower pressures & better structural stability = longer life
• Particle size
- Smaller particle = higher operating pressure = higher efficiency

Detectors

UV spectrophotometer is used consist of a flow cell placed at end of column to receive portion of eluted solute.
Light of specific wavelength pass through the cell, some parts are absorbed, the others are transmitted
Specific sample has high absorbance to specific wavelength
Amount of absorbed light (A) is proportional to the
concentration of sample
A = constant X b X c

UV detector

Types of UV detectors

There are several types of UV detectors:
1- detectors operating at fixed wavelength [= 254 nm] emitted by low-pressure Hg lump].
2- detectors operating at variable l depending on lmax of the components leading to increased sensitivity [from deuterium or xenon lamp with monochromator or filter to generate monochromatic radiation at selected l].
3- Multi-wavelength detectors : measure at 2 or more selected l at the same time.

Other detectors

Refractometer ,
Fluorimetric.
Potentiometric.
Voltametric.
Polarographic or electrochemical detectors

Data collection

Data collection:
when a solute in eluted will give an absorption peak,its height or area is corresponding to the concentration of solute.
Old manual detectors: give printout of peaks or peak areas.
New detectors: provide chromatograms with accompanied peak areas & heights.

Retention time

Retention time “ t ” : time spent from injection till appearance of tip of the peak
Retention time is characteristic for each compound so a standard of the analyte must be injected to assure retention time of the substance.

Data collection

Peak area = h X W/2 = h X W h/2
Where:
h = height of the peak.
W = width at peak base line.
W h/2 = width at half height of the peak.

Capacity Factor

VR = Retention volume
V0 = Retention volume of solvent
t R = Retention time
t 0 = Retention time of solvent
t’R = Actual retention time

Selectivity

Column Efficiency

Resolution

Quantitative HPLC

Automatic injectors [Auto samples]	Manual injectors [a syringe]
increases degree of accuracy in volume injected	decreases degree of accuracy in volume injected
Use external standardto construct calibration curve to determine concentration.	Use internal standard to cancel error which may occur during manual
External standard: ·Injectedseparately in each run. ·Of the same substance of analyte. ·comparepeak response [height or area].	Internal standard: ·Injected with fixed concentration together with test or standard solution. ·Of different substance than analyte. ·Mustnot interfere with peaks measured. ·We compare peak response ratio.

Ideal chromatogram

1.Peaks should be sharp & symmetric.
2. Peaks should be well separated from one another (each peak should start at the baseline and comes back down to the baseline).
3.The base line should be straight (with no drifting up or down & no underlying impurities).
4.The base line should be free from background or electric noise.
5.The chromatographic run time should be reasonable (15 minutes is ideal).
6.The developed procedure should have good sensitivity. When the analysis is repeated under the same conditions, the chromatogram should be reproducible

Esam Othman

Hanaa Hashem

1-Modes of separation in HPLC

2008-04-05T10:32:00.000-07:00

There are theree main charactersitics of any organic compound which are used as the basis of separation in chromatography. These are:
• Polarity
• Electrical Charge
• Molecular Size
We are only going to be concerned with separation based on polarity since it is the most commonly used for chromatographic separation.

Separations Based on Polarity:
Organic molecules are sorted into classes according to the principal functional group(s) present in the molecule. Using a separation mode based on polarity , the relative degree of retention of different kinds of molecules is mainly determined by the nature and location of these functional groups.Classes of molecules can be arranged into a range of chromatographic polarity from highly polar to highly non-polar as shown below.

Polarity- based chromatography follows the rules which govern the partition of organic compounds between oil and water and which determines the partition coeffecient of a compound. In other words, water, being a small molecule with a high dipole moment, is a polar compound. On the other hand, benzene which is an aromatic hydrocarbon, is a non-polar compound. Molecules with similar chromatographic polarity tend to be attracted to each other and those with dissimilar polarity exhibit much weaker attraction and may even repel one another. This is the main frame for chromatographic separation modes based on polarity. Another way to think of this is : oil (non-polar) and water (polar) don’t mix. Chromatographic separations based on polarity depends upon the stronger attraction between likes and the weaker attraction between opposites. Remember, “Like attracts like” in polarity-based chromatography (the opposite of magnitism).

Based on the discussion above, in order to design a chromatographic separation system we create competition for the various components of the mixture between a mobile phase and a stationary phase with different polarities (thus forcing the different commponents to partition between the two pahses as they go through the column). Then, compounds in the sample that are similar in polarity to the stationary phase (column packing material) will be delayed because they are more strongly attracted to the particles. Compounds whose polarity is similar to that of the mobile phase will be preferentially attracted to it and move faster. Thus, the difference in the force of attraction of the different components of the mixture to the stationary and mobile phase causes separation between the analytes by changing the speed by which each analyte moves through the column. The larger the difference (between the components of the mixtrue) in the speed of movement the higher the selectivity of the chromatographic system.
Polarity of the mobile phase ranges between non polar molecules presented by hexane to the most polar molecule (water). Between the two solvents lies a range of molecules with varying degree of polarity as follows: water being most polar followed by the less polar methanol then acetonitrile, tetrahydrofuran then the least polar Hexane. We can always create mixtures with polarity that lies in between two solvents for example a mixture of water and methanol makes a solven mixture which is more polar then methanol and less polar than water. How much less polar than water will depend on the percentage of methanol in the mixture.When the analyte mixture is introduced on the column they become attracted to the particles of the stationary phase. A mobile phase which more effectively attracts the analyte will compete for it and displaces it from the stationary phase causing the analyte to move faster through the column (weakly retained).

For stationary phases which make the packing material of the column. Silica is the most polar, hydrophilic (water-loving) surface containing acidic Silanol ( silicon containing analog of alcohol) functional group. The activity or polarity of the silica surface may be modified selectively by chemically bonding to it less polar functional groups [bonded phase]. For examples the binding of cyanopropylsilyl- [CN] will give a less polar stationary phase then the less polar n-octylsilyl- [C8] then n-octadecylsilyl- [C18, ODS] mieties . The latter is a hydrophobic [water-hating], very non-polar packing.
After considering the polarity of both phases, then, a chromatographer must choose a mobile phase which will allow the analytes of interest to be retained by the stationary phase, to a certain extent, but not so strongly that they cannot be eluted. Among solvents of similar strength, the chromatographer considers which phase combination may best exploits of the little differences in polarity and solubility of the components of the mixture to maximize the selectivity ( separation between the eluted peaks) of the chromatographic system. But, as you probably can imagine from the discussion so far, creating a separation based upon polarity involves knowledge of the sample, and experience with various kinds of analytes and retention modes. Thus the chromatographer will choose the best combination of a mobile phase and stationary phase with sufficiently different polarities. Then, as the mixture of analytes moves through the column, the rule like attracts like will determine which analytes slow down and which move at a faster speed.

Normal-Phase HPLC :
A mixture of compounds was analized using a column packed with seilica (polar stationary phase) as shown above with a much less polar [non-polar] mobile phase. This mode of chromatography is known as normal phase.
The mixture is composed of three comounds. Compound A(yellow) which is very polar, Compound B (red) and is less polar and compound C (blue) is the least polar. SInce the stationary phase (silica) is polar thus it retains the polar yellow component most strongly. The relatively non-polar blue compound moves fastest down the column since it is the least polar so is the most attracted or retained by the mobile phase, a non-polar solvent, and elutes (comes out of the column) quickly.

Reversed-Phase HPLC:

The term reversed-phase describes the chromatography mode that is just the opposite of normal phase, namely the use of a polar mobile phase and a non-polar (hydrophobic) stationary phase. the figure above shows the same mixture of drugs separated before and which is composed of compound A (yellow) which is the most polar, compound B (red) of intermediate polarityand compound C (blue) which is the least polar in the mixture.
Now the most strongly retained compound is the more non-polar compound C (blue) , since it is the most attracted to the non-polar stationary phase. The polar yellow compound A, being weakly retained on the non polar stationary phase and is the nost attracted by the polar mobile phase, thus moves the fastest through the column packing, and elutes earliest (comes out of the column first) .
Reversed-phase chromatography is now concidered the most popular pahse of separation due to its higher reproducibility and ease of applicarion to a wide range of compounds.

Now as each band (separated compund) is eleminated from the column it passes through the detector'e cell and gives a responce proportional to its concentration. The recorder will record a peak at a time corresponding to the time at which the particular band comes out (eluted) and the integrator will calculate the area or the hight of the peak which, in turn, is relative to the concentration of the band. Thus giving the known form of a chromatogram which is a plot of retention time vs. peak hight or area. In the case of reversed phase elemination of the yellow (1), red (2) and blue (3) compounds will give three well resolved peakson the chromatogram to the right.
Given all the above facts and knowing the nature of the sample mixture being used the chromatographer is always facing the challenge of producing a chromatographic separation method which best suits his purposes. The chromatographer is always aiming at a chromatographic system which will produce an ideal chromatogram that will help him reproducibly analize his mixture.

References:
1-Laboratory outline and notebook for Pharamceutical Chemistry; Fourth year (802).
2-Instrumental methods of chemical analysis, Glen W. Ewing (1998).
3-Contemporary Instrumental analysis, Kennetha A. Rubinson and Sudith F. Rubinson.
4-Waters "the science of what is possible".
5-HPLC and CE principles and practice, Andrea Wetson.

2-The development of a good chromatographic separation

2008-04-05T06:32:00.000-07:00

Ideally a good chromatogram should have the following charecterstics:

1- The peaks should be sharp and symmetric.
2- Peaks should be well separated from one another ( each peak should srtart at the baseline and comes back down to the baseline).
3- The base line should be straight ( with no drifting up or down and no underlying impurities).
4- The base line should be free from background or electric noise. (What is the difference between 3 and 4).
5-The chromatographic run time should be reasonable (15 minutes is ideal).
6-The developed procedure should have good sensitivity.
7-When the analysis is repeated under the same conditions, the chromatogram should be reproducible.

A good chromatogram may look like one of the following examples:

Chromatogram (1)

Chromatogram (2)

Chromatogram(4)

Chromatogram (3)

The peaks in all chromatograms are well separated, sharp and they all come back down to the base line. There are no underlying impurity, no drifts and no noise in the base line. The analysis time is reasonable. In chromatogram (2), the analysis time is a little longer than would be concidered optimal but the number of components to be separated are large and the good resolution between the peaks makes up for the slightly longer time of chromatographic analysis. However, obtaining an ideal chromatogram like one of the above is only acheived after an effort. Although knowlege of the nature of the mixture to be analized and experience in chromatography may help us arrive at a good combination of stationary and mobile phase that produces a reasonable chromatogram. Some times highly unacceptable chromatograms like the ones below may be obtained .

In this chromatogram although the chromatographing time is reasonable, peak 2 and 3 are not well resolved. peak 1 seams to have another peak underneath it causing a shouder (lack of symmetry) after the epak.

In this chromatogram resolution is very bad. The baseline is drifting upwords. This would not allow for proper calculation of peak area or hight.

Some times slight modification of the mobile phase will improve the separation and in other cases the chromatgraphic separation may need a drastic change in the mobile pase or the type of the stationary phase which may even alter the sequence of elution. Study the following examples.

(B)
The two achromatograms (A) and (B) are of the same mixture of two components. The mobile ohase used to develop chromatogram (A) did not lead to complete separation of the two components. Therefore chaniging the mobile phase lead to much better separation in the above chromatogram(B).

(A)

The same argument holds for the mixture below:

Chromatogram(A)

Chromatogram (B)

In the above chromatograms complete resolution was not acheived by using the mobile phase in chromatogram(A). While the mobile phase in (B) was very successful in providing complete separation of the peaks in the mixture.

(B)
Now in this example a very good resolution was obtained using mobile phase (A) changing the mobile phase in (B) only increased the run time. Therefore there was no need to change chromatographic conditions.

(A)

{A} In the above chromatogram (A)although the peaks seam well separated peak 7, 6 are eluted logether. This may indicate that they are very close in polarity.
{B} In the second chromatogram a different mixture of solvents was used as a mobile phase with completely different polarity. The result was a much better chromatogram, well resoved peaks but, on the other hand the order of elution of the peaks was changed.

Now what about the mixtures given below?

...........II........................III

......I

3-problems encountered during the analysis, their causes and solution

2008-04-05T05:23:00.000-07:00

Now a chromatographic method ( the appropriate combination of stationary phase,and flow rate) which gives us a satisfactory chromatogram has been chosen. Several samples of the same mixture were analized and a reproducible pattern was obtained. The analysis should be running smoothely afterwords to enable us to construct a calibration curve and analize our unknown samples. Sometimes, however, we may run into unexpected problems (changes in performance of the instrument or changes in the chromatographic pattern that has been reproducible thus far etc...) which should not normally happen. Finding out the causes of the problem and fixing them is a valuable experience a good chroamtogrammer should acquire. There are several books and manuals dealing with the subject (truoble shooting). However, here are some of the problems we commonly face, their possible causes and solitions.

I- No peaks or very small peaks

Chromatogram(A)

Chromatogram(B)

The two chromatograms given above show the normally produced chromatogram (A) which was a good chromatogram then we run into a problem where we see no peaks coming out of the colum. A couple of causes (problems) might have arrised which lead to this.

Proble1em(1)

No peaks or very small peaks
Possible cause solution
Detector is off ( forgot to turn on the
detector).
Check detector
Broken connections to recorderelectric connections theat carry the signals to the detector and from the detector to the recorder are broken).
Check detector connections
No sample/Wrong sample
Check sample. Be sure it is not deteriorated. Check for bubbles in the
vials
Wrong settings on recorder or
detector
Check wavelength setting (wrong wavelength) and sensitivity
setting

Problem (2)

No Flow

Possible cause solution
a-Pump off
Start Pump
b-Flow interrupted
Check reservoirs. Check position of the inlet tubing. Check loop for obstruction or air. Check degassing of mobile phase. Check compatibility of the mobile phase components
c-Leak
Check fittings. Check pump for leaks and precipitates. Check pump seals.

d-Air trapped in the
sys
Disconnect column and prime pump. Flush system with 100% methanol or
isopropanol

II-CHanges in pressure

Problem (3)

Decreasing Pressure( no pressure or lower than usual
Possible cause solution
Insufficient flow from pump
Loosen cap on mobile phase reservior (tigt capping of the mobile phase container while the pump is pulling solven at high rate may create vacume thus preventing the pump from pulling out the right ammount of solvent).
Leak in hydraulic lines from pump to column.
Check reservoirs. Check position of the inlet tubing. Check loop for obstruction or air. Check degassing of mobile phase. Check compatibility of the mobile phase components.
Leak
Check fittings. Check pump for leaks and precipitates. Check pump seals
Air trapped in the system
Disconnect column and prime pump. Flush system with 100% methanol or isopropanol

Problem (4)

*Increased pressure(pressure higher than usual)*	*Possible cause*	*solution*
	Column blocked with irreversibly adsorbed sample	*Improve sample cleanup; use guard column; reverse-flush column with strong solvent to dissolve blockage.*
	*Microbial growth*	*Use at least 10% organic modifier in mobile phase; use fresh buffer daily; add 0.02% sodium azide to aqueous mobile phase; store column in at least 25% organic solvent without buffer.*
	*Salt precipitation (especially in reversed-phase chromatography with high concentration of organic solvent in mobile phase).*	Ensure mobile phase compatibility with buffer concentration; decrease ionic Ensure mobile phase compatibility with buffer concentration; decrease ionic Check fittings. Check pump for leaks and precipitates. Check pump seals
	*The pressure comes back to normal when the injector is disconnected from column = blockage in injector.*	Clean the injector
	Blocked column frit.	*1. Backflush column (run the solvent opposite to the normal direction of flow (if permitted)).* 2-Replace the frie

III-Change in the chromatogram

a-Problem(5)

Variable retention time (Change in retention time)

Retention time of the different peaks of the mixture sgould be reproducible as long as there was no change in the chromatographic conditions (mobile phase, stationary phase or flow rate). In fact some times retention time is used as a confirmation of the structure of an unknown peaks.In the given chromatograms, the three peaks come out at 1.5, 4 and 7 minutes respectively. after several reproducible runs, without intentional changes in conditions, the peaks started coming out farther apart at 6, 10 and 15.

Variable retention time (Change in retention time)

Possible cause solution
Contamination buildup
Flush column occasionally with strong solvent.
Selective evaporation of mobile-phase component.
Cover solvent reservoirs; use less-vigorous helium purging; prepare fresh mobile phase.
Loss of bonded stationary phase.(Column aging)
Some times adjusting pH of mobile between 2 and 8 may help. Or change the column.
b-problem(6)

peak tailing

Ipeak tailing
Possible cause solution
a-Blocked frit
1. Reverse flush column (if allowed)2. Replace inlet frit.
b-( not all peaks ar tailing) interfering peak(s)
1. Use longer column 2. Change mobile-phase and/or column (selectivity)
c-Something wrong with the mobile-phase pH
1. Adjust pH 2. adjust components of mobile phase.
d-Sample reacting with active
sites.*
1.Add ion pairing reagent (an ion that forms a pair with the compound being eluted thus preventing it from reacting with the active sites on the selica).
2.Change the column.
*explanation: when the column is used for a long time some of the chemically bonded substances (e.g. C18) may be depleted exposing active groups on the selica (aging) which bind strongly to some of the components of the mixture causing them to be eluted with difficulty and thus dragging behind the main bulk of the peak causing a tail formation.
problem (7)

Split Peaks

Split Peaks
Possible cause solution
a-Contamination on guard or analytical column inlet.
1. Remove guard column and attempt the analysis 2.Replace guard column if necessary 3.If analytical column is obstructed, reverse and flush.
4.If the problem persists, then the inlet is probably clogged. Change frit.
b-Sample solvent incompatible with mobile phase
Change solvent; and whenever possible, inject samples in mobile phase
problem (8)

The appearance of extra peak (s)
Possible cause solution
a-contaminating components in the sample
clean up the sample.
b-Late eluting peak(s) :peaks which did not come out during the previous analysis and started coming out after the next sample was injected.
Increase run time or gradient slope (increasing gradient slope leads to faster change in solvent composition and therefore better resolution).

problem(9)

Base Line drift

Base Line drift
Possible cause solution
a-Mobile phase contaminated, deteriorated, or prepared from low-quality materials.
Use HPLC-grade solvents, high-purity salts, and additives.
b-Contaminant or air buildup in detector cell.
Flush the cell with methanol or other strong solvent.
c-Mobile-phase mixing problem or change in flowrate.
Correct composition of the mobile phase or flow rate (to avoid running into such a problem, routinely monitor composition and flow rate).
d-Slow column equilibration, especially when changing mobile phase
Run 10–20 column volumes of new mobile phase before analysis

problem (10)

Baseline Noise

Baseline
Noise
Possible cause solution
a-Air in mobile phase, detector cell, or pump.
1. Degas mobile phase
2. Flush to remove air from detector cell or pump.
b-Other electronic equipment on the same line.
Isolate detector, or recorder to determine if the source of problem is external; correct as necessary.
c-Pump pulsation.
Incorporate pulse dampener into system.
problem (11)
broad peaks

broad peaks
Possible cause solution
a-Mobile-phase composition
changed.
Prepare new mobile phase.
b-Mobile-phase flow rate too low.
Adjust flow rate.
c-Leaks (especially between column and detector)
1. Check for loose fittings
2. Check pump for leaks, salt build-up.
3. Change seals if necessary.
d-Column overloaded
Inject smaller sample on the column (e.g., 10 µl vs.100 µl) or 1:10 and 1:100 dilutions of sample.
e-tubing between column and detector too long or ID (internal diameter) too large
use shorter piece of tubing or tubing with smaller ID
f- guard column contaminated or worn out.
replace guard
column.
g-Column contaminated or worn out
flush old column with strong solvent or replace the old column with a new one.
h- peaks represent two or more poorly resolved compounds (loss of resolution)
see the next problem.
problem(12)
Loss of resolution
loss of resolution
Possible cause solution
a-mobile phase contaminated or deteriorated (causing retention times to change)
prepare new mobile phase.
b-obstruction of gaurd or analytical column.
1- remove gaurd column and attempt the analysis and replace the gaurd column if necessary
2- if the analytical column is obstructed reverse and flush if the problem pesists then the column may be fouled with strongly retained contaminant or the inlet may be blocked then replace the frit or replace the analytical column
c-Aging or deteriorated column (will lead to loss of selectivity)
Replace the column with a new
one

*No peaks or very small peaks*	*Possible cause*	*solution*
	Detector is off ( forgot to turn on the detector).	Check detector
	Broken connections to recorderelectric connections theat carry the signals to the detector and from the detector to the recorder are broken).	Check detector connections
	No sample/Wrong sample	Check sample. Be sure it is not deteriorated. Check for bubbles in the vials
	Wrong settings on recorder or detector	Check wavelength setting (wrong wavelength) and sensitivity setting

*No Flow*	*Possible cause*	*solution*
	*a-Pump off*	*Start Pump*
	*b-Flow interrupted*	*Check reservoirs. Check position of the inlet tubing. Check loop for obstruction or air. Check degassing of mobile phase. Check compatibility of the mobile phase components*
	*c-Leak*	*Check fittings. Check pump for leaks and precipitates. Check pump seals.*
	d-Air trapped in the sys	Disconnect column and prime pump. Flush system with 100% methanol or isopropanol

*Ipeak tailing*	*Possible cause*	*solution*
	a-*Blocked frit*	*1. Reverse flush column (if allowed)2. Replace inlet frit.*
	b-( not all peaks ar tailing) interfering peak(s)	1. Use longer column 2. Change mobile-phase and/or column (selectivity)
	c-Something wrong with the mobile-phase pH	1. Adjust pH 2. adjust components of mobile phase.
	d-Sample reacting with active sites.*	1.Add ion pairing reagent (an ion that forms a pair with the compound being eluted thus preventing it from reacting with the active sites on the selica). 2.Change the column.

*Base Line drift*	*Possible cause*	*solution*
	a-Mobile phase contaminated, deteriorated, or prepared from low-quality materials.	Use HPLC-grade solvents, high-purity salts, and additives.
	b-Contaminant or air buildup in detector cell.	Flush the cell with methanol or other strong solvent.
	c-Mobile-phase mixing problem or change in flowrate.	Correct composition of the mobile phase or flow rate (to avoid running into such a problem, routinely monitor composition and flow rate).
	d-Slow column equilibration, especially when changing mobile phase	Run 10–20 column volumes of new mobile phase before analysis

*Baseline* *Noise*	*Possible cause*	*solution*
	a-Air in mobile phase, detector cell, or pump.	1. Degas mobile phase 2. Flush to remove air from detector cell or pump.
	b-Other electronic equipment on the same line.	Isolate detector, or recorder to determine if the source of problem is external; correct as necessary.
	c-Pump pulsation.	Incorporate pulse dampener into system.

*broad peaks*	*Possible cause*	*solution*
	a-Mobile-phase composition changed.	Prepare new mobile phase.
	b-Mobile-phase flow rate too low.	Adjust flow rate.
	c-Leaks (especially between column and detector)	1. Check for loose fittings 2. Check pump for leaks, salt build-up. 3. Change seals if necessary.
	d-Column overloaded	Inject smaller sample on the column (e.g., 10 µl vs.100 µl) or 1:10 and 1:100 dilutions of sample.
	e-tubing between column and detector too long or ID (internal diameter) too large	use shorter piece of tubing or tubing with smaller ID
	f- guard column contaminated or worn out.	replace guard column.
	g-Column contaminated or worn out	flush old column with strong solvent or replace the old column with a new one.
	h- peaks represent two or more poorly resolved compounds (loss of resolution)	see the next problem.

*loss of resolution*	*Possible cause*	*solution*
	a-mobile phase contaminated or deteriorated (causing retention times to change)	prepare new mobile phase.
	b-obstruction of gaurd or analytical column.	1- remove gaurd column and attempt the analysis and replace the gaurd column if necessary 2- if the analytical column is obstructed reverse and flush if the problem pesists then the column may be fouled with strongly retained contaminant or the inlet may be blocked then replace the frit or replace the analytical column
	c-Aging or deteriorated column (will lead to loss of selectivity)	Replace the column with a new one