1-Modes of separation in HPLC

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

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

IV

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

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