HPLC A Praactical User''''S Guide Part 4 pdf

Increasing the polarity difference between the stationary and mobile phases increases the retention of compounds with polarities most like the column.. For example, for a polar silica co

times 2-fold while increasing efficiency only by 1.4-fold due to increased diffusion

Finally, we have variables affecting efficiency that can be controlled at the time of the run These are pump flow rate, extracolumn volumes in the instru-ment used, and the method of calculation Flow rate is the major efficiency variable that I use during methods development Generally, halving the flow rate will increase separation around 40% I do much of my scouting at 2.0 mL/min, knowing that I can improve separation by dropping to 1.0 mL/min Plotting of efficiency versus flow rate shows that each diameter of packing has its own optimum flow rate Efficiency decreases at higher flow rates In the microparticulate packings, large packing diameters show a more rapid loss of efficiency with increasing flow rate than do smaller packings

Decreasing extracolumn volumes is critical to HPLC success The most important volumes are those immediately adjacent to the column: zero-dead-volume end-fittings, inlet and outlet tubing diameters, and detector cell volumes From the time the sample enters the injector until it exits the detec-tor, nothing must add increased mixing space Tubing from injector to column must be 0.010 in for 5-mm and 10-mm packings with tubing lengths no more than 4–6 in for the 5-mm Use 0.007-in tubing about 3 in long or less for 3-mm packing Zero-dead-volume endcaps and connectors must be prepared cor-rectly, so that tubing ends butt firmly against the fitting We covered the prepa-ration of compression fittings in Chapter 3, but if you find efficiency drops after you change a fitting, check the dead-volume fit For detector cells, the rule of thumb is 8–12 mL; anything larger acts increasingly as a mixer for your already separated bands

Tubing volumes outside the critical injector-detector range are important only if you are doing recycling or collecting samples Pump-to-injector tubing

is generally 0.020-in; vents, flush valve, etc may use 0.04-in Be sure you know what these look like and do not confuse them with injector tubing In telling tubing apart, 0.02-in and 0.01-in are the most difficult to tell apart If you have

to look twice to make sure there really is a hole, it is probably 0.01-in If you are in doubt, put them next to each other By comparison, 0.04-in tubing looks like a sewer pipe

There are many methods used to calculate efficiency All methods give the same results with ideal, Gaussian peaks Real chromatography peaks tend

to tail on the backside of the peak (away from the injection mark on the

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Table 4.1 Relationship of efficiency to flow rate

Efficiency Changes with Particle Size Packing diameter (mm) Plates/meter Flow rate (mL/min)

chromatogram) When column problems occur they often tend to show up as increased tailing Calculation methods that use a peak width high on the peak miss these changes and give artificially high efficiencies The 5s method described above is excellent for detecting early appearance of tailing If you’re planning on using a calculation using half-peak width, make sure there is some method of measuring and correcting for peak asymmetry

The retention factor, k′, also called the capacity factor, is the usual starting point for methods development The retention factor, as its name implies, is

basically a measure of how long each compound stays on the column; V Oused

to determine k′is usually only roughly measured; k′is a simply a multiple of

the V Odistance (see Fig 4.5)

The major usable variable controlling k′is solvent polarity While temper-ature and column polarity also effect retention times, they do not show the same direct, linear relationship for all peaks and are usually classed under the separation factor (a)

Increasing the polarity difference between the stationary and mobile phases increases the retention of compounds with polarities most like the column Compounds stick tighter and peaks will broaden through diffusion Decreas-ing the polarity difference will make thDecreas-ings come off faster and shoved together Peaks will be less resolved and sharper

For example, for a polar silica column equilibrated with a mobile phase of methylene chloride in hexane (nonpolar), you would dilute with more hexane

to increase the k′of relatively polar components Adding methylene chloride,

the more polar of the two solvents, would decrease k′s causing all components

to wash off faster With k′changes, peak position changes are proportional and

in the same direction The order of resolved peaks will remain the same; unre-solved peaks should begin to pull apart

If in our model system, we had used 80% methylene chloride/hexane and the red peak had partially overlapped the backside of the blue peak, we would attempt to resolve it by reequilibrating in 40% methylene chloride/hexane and reinjecting We could expect that we should see two well-resolved peaks; if not,

we could go to a 20% mixture More than likely, we would have overshot on the first change and would have to fine-tune back toward the 80% mixture Simply by modifying the solvent polarity, we are able to increase or decrease

k′ and contract or spread our separation This k′ development is our usual starting point in methods development

So far, I have referred only to “normal-phase” separations on polar columns However, around 80% of the separations in the literature are made

on “reversed-phase” columns To understand these terms, we need a little history

Figure 4.5 Retention factor equation.

Early “high-pressure” packings were cross-linked ion exchange resins and polymeric size separation, gel permeation packings The first high-pressure columns for partition separations were packed with the same material as is used in open columns or for preparing TLC plates These were 35- to 60-mm diameter silica, a very polar packing material To achieve separation, nonpolar solvents were used for elutions These solvents were flammable, volatile, toxic,

or expensive After a few years, someone decided to coat the silica with non-polar compounds similar to those used in GLC column so that non-polar solvents, such as water, could be used for elution The problem with these coatings was that they tended to wash out with the mobile phase, bleed into the detector, and contaminate the collected sample

This was overcome by chemically bonding the coating to silica leading to the first “abnormal” packing materials Because these packings could be run

in aqueous solvents and did not require the careful drying and handling of the normal-phase columns, they quickly became very popular Since no one wanted to admit to being an “abnormal” chromatographer, when they reached the majority they quickly renamed themselves “reversed-phase” chromatographers

The first of the really successful coatings was a long-chain, saturated hydrocarbon with 18 carbons These octadecyl- (ODS), RP18, or C18columns are still the most commonly used HPLC columns, primarily because of the versatility they have shown Other packing materials have appeared with shorter or longer side-chains, and, with a variety of functional groups on the side-chains, greatly extended the possible separations that can be achieved with HPLC

Retention changes work exactly the same with reverse-phase column as with normal-phase columns Increasing the polarity difference between

column and mobile phase increases the k′s of the components However, since the column is nonpolar, we now must add more of the polar solvent to make compounds stick tighter On our reversed-phase column, our dye mixture would also elute in opposite order, the more polar red dye would have less affinity for the nonpolar column and would elute before the nonpolar blue dye By controlling the column nature, you control the elution order Figure 4.6 illustrates the effect of solvent polarity changes on a separation

As we mentioned earlier, there is a limit to usefulness of k′changes Because

it is a convergent term in the resolution equation, the larger the value of k′,

the less the effect a polarity change has on Rs Beyond k′ =8–10, changing k′ has only a negligible effect, except on run time At this point, the next step is

to change resolution, Rs, by using the separation factor, a.

The separation factor, a (Fig 4.7) is calculated by dividing the k′s for the two peaks under question It measures the separation between the two peak centers Components with an a = 1.0 overlap completely; beyond a = 2.0,

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compounds can be separated by separatory funnel Large as are needed in HPLC only for preparative runs

When we change retention with solvent polarity, all peaks show an equiv-alent shifting in the same direction A variable producing an a change causes relative peak positions to shift; individual peaks exhibit different amounts of

shift, both in size and direction Thus, k′changes spread separations already present; with a changes new separations are created With an a change, rela-tive peak positions can even reverse

Temperature is the first of the variables affecting separation Increased tem-perature decreases retention time on the column, sharpens peaks, and pro-duces the change in relative peak retentions typical of an a effect At first, this appears to be the ideal variable, similar to temperature programming for GLC However, temperature changes have some drawbacks

First, temperature is generally limited to an effective range of 20–60°C by solvent vapor pressures Higher temperatures can vaporize solvent in the column leading to column voiding and cavitation, similar to a vapor lock in a car’s engine on a hot day It can produce chemical changes in some compounds being separated, catalyzed by contact with the hot, acidic silica surface Even more important is the effect temperature has on the column packing Bonded phase columns are prepared by chemically bonding an alkyl chlorosilane to the oxygen on the silica This process can be reversed by hydrolysis, especially under acidic conditions, leading to bonded-phase bleeding and column per-formance changes Heat accelerates the process If you’re only getting 3 mo life from your columns, this might not be an important consideration But, one of the goals of this text is to show you how to extend column life

Figure 4.6 Effect of polarity changes.

Figure 4.7 Separation factor equation.

Recent changes in column stability with zirconium-based and hybrid silica columns have lead to resurgence in the use of column jackets to elevated temperature to speed analysis time The problem of sample degradation at these higher temperatures remains a continuing problem as it does in GC separations

The separation factor (a) is also referred to as the chemistry factor It can

be modified by changes in the chemistry of the components that make up the chromatographic system: column, solvent, and sample Changing the column surface chemistry from the very nonpolar C18 to C8obviously increases the column polarity as the compounds are drawn closer to the silica surface We would predict that nonpolar compounds would elute faster, and so they do However, observation of the peaks shows peak shifting typical of an a vari-able If we substitute a phenylethyl group for octyl, we maintain the same polarity, but now we see dramatic changes in selectivity The so-called phenyl column has an affinity for aromatics and double bonds It will separate fatty acids on the basis of the number of double bonds as well as chain length Octyl columns separate only on chain length differences

The most common variable used to control a is the “stronger” solvent in the mobile phase The stronger solvent is the mobile phase component most like the column in polarity Changing the chemical nature of this stronger solvent will produce shifts in the relative peak positions For instance, if we are unable to achieve the desired separation on a C18column using acetonitrile in water, we can produce an a effect by shifting to methanol in water: an oppo-site effect occurs on switching to tetrahydrofuran in water (Fig 4.8)

This is true even if we adjust the polarity of the new mixtures to match that

of the previous mobile phase We can produce other a changes by adding mobile phase modifiers to our solvents Buffers, chelators, ion pairing reagents, and organic modifiers can all be used to change or fine-tune the separation

We will cover use of all of these in detail in a Chapter 7

The final a modifier, preparing derivatives of a mixture, is our court of last

resort If two compounds cannot be separated by changing N, k′, or the

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Figure 4.8 Effect of “stronger” solvent changes.

chemistry of the column or mobile phase, then changing their chemical nature

by making derivatives should lead to compounds that can be separated We use this only as a last gasp separation technique Usually, we can separate most compounds directly Derivatives are more commonly used in HPLC to change

a mixture’s solubility or to produce compounds with strong extinction coeffi-cients to increase detection sensitivity

So far, we have dealt only with partition chromatography in which compounds equilibrate between the mobile phase and the column based on differences in their polarity Ion-exchange chromatography uses the type and degree of ion-ization of the column and compounds to achieve a separation Here, opposites rather than likes attract; compounds with charges opposite to that on the columns are attracted and held by the column Elution is achieved by com-petitive displacement; an excess of an ion with the same charge as the bound compound pushes it off the column The tighter the ionic bonding to the column, the longer the compound stays on the column

Ion-exchange columns are made of a number of backbone materials: silica and zirconium, like the reverse phase columns, and heavily cross-linked, organic polymers Bound to these are organic bonded phases containing func-tional groups that either have permanent ionic charges or in which ionic charges can be induced with pH changes

Two warnings about using polymeric columns: Early polymeric column for

ion exchange would not tolerate much pressure or organic solvents Recent columns are more heavily cross-linked and show more pressure tolerance, but

be sure to check the manufacturer’s column shipping notes for use limitations Few will tolerate pressures above 2,000 psi without collapsing Some organic solvents can cause the column bed to swell or shrink on changing solvents, which can lead to bed collapse or voiding

Charged functional groups, which give these columns their separating char-acter, are of two types: anionic and cationic Anionic packing materials have

an affinity for anions (negatively charged ions) and have positively charged functional groups on their surfaces, usually organic amines Cationic packings attract cations (positive charges) with negative functionalities, usually organic acids and sulfonates Cationic and anionic olumns can both be subdivided into either strong or weak types Strong columns have functional groups that possess either permanent charges (i.e., quaternary amines) or have charges present through the full pH range used for HPLC (i.e., sulfonic acids) Weak columns have function groups with inducible charges At one pH they are uncharged, at a different pH they are charged Examples are organic acids, which are uncharged at pH 2.0, but form cations at pH 6.5, and organic primary amines, which are positively charged below pH 8.0, but exist in the free amine form above pH 12

Let us examine a silica-based cationic (sulfonate) ion exchange separation (Fig 4.9) The column is equilibrated in 50 mM sodium acetate An injection

of amines and an alcohol in the mobile phase is made The same mobile phase,

or one containing increased amounts of sodium acetate, is used to elute fractions

The alcohol will come off in the void volume of the column since it has no attraction to the column The amines will be retained, because at the pH of the acetate solution they are protonated and have a positive charge As more mobile phase passes the through the column, its sodium ions begin to compete for the sulfonate sites with the bound amines Through a mass effect, the amines are displaced down the column until, finally, they elute into the detector The amine that has the strongest charge and binds the tightest is eluted last

The first commercial HPLC system was sold to do gel permeation (GPC) or size separation chromatography It is the simplest type of chromatography, the-oretically involving a pure mechanical separation based on molecular size

SIZE EXCLUSION CHROMATOGRAPHY 57

Figure 4.9 Cationic-exchange separation model.

The column packing material surface is visualized as beads containing tapered pits or pores As the mobile phase sweeps the injection passed these pits, the dissolved compounds penetrate, if their largest diameter (Stokes radius) is small enough to fit (Fig 4.10) If not, they wash down the column with the injection front and elute as a peak at the column void volume, which

is called the exclusion volume.

Returning to the compounds that entered the pit, we find that large parti-cle can not penetrate as deeply down the pore as can smaller compounds The smaller the diameter, the deeper the penetration, and the longer the com-pound takes to elute.The largest comcom-pounds wash out quicker, follow a shorter path, and elute just later than the totally excluded compounds Traveling down the column, these resolving compounds wash in and out of many pores mag-nifying the resolution achieved by differences in the path lengths they follow Finally, we reach a point where all compounds of a certain diameter or smaller reach the pore bottom, wash out, and elute in a single peak This is referred

Figure 4.10 Size-separation model.

to as the inclusion volume If the exclusion volume is found at V O, the

inclu-sion volume appears at approximately 2 Vo.

From this, we can see we have three types of peaks: 1) the exclusion peak, containing all molecules of a certain size or larger; 2) resolved peaks of inter-mediate diameter; and 3) the inclusion peak containing all compounds of a given diameter and smaller In a crude mixture of compounds, we are forced

to suspect that both the exclusion and inclusion peaks contain multiple components

Just as it is possible to prepare a column with a single pore size, It is possi-ble to prepare columns with differing pore size Each would have its own par-ticular ratio of exclusion/inclusion diameters A column bank of columns containing different pore packing can be used to separate a mixture with a wide range of compound sizes Columns of varying exclusion/inclusion limits can be connected with the smallest exclusion limit column first in the series

If the columns are selected so the first’s exclusion limit overlaps the second’s inclusion limit and so forth, the column bank produced has the first column’s inclusion limit and the last column’s exclusion limit.Again, remember the pres-sure problem when stacking columns; prespres-sure increases proportionally to the number of columns You may have to run very slowly if you are using pres-sure fragile columns

GPC columns are referred to as molecular weight columns, but they actu-ally separate molecules according to their largest dimension True molecular weight measurements would be independent of shape As long as we work with simple, spherical compounds, there is a direct relation between exclusion volume and molecular weight within the resolved range Columns can be cal-ibrated with standards of known molecular weight and used for molecular weight determinations These measurements break down at higher molecular weights with compounds with nonspherical shapes (i.e., proteins), which change shape and apparent size with changes in the mobile phase Solvent con-ditions that force all molecules into long, rigid shapes aid in molecular weight determinations (i.e., 0.1% sodium dodecyl sulfate [SDS] is used for protein molecular weights)

Size separation columns are available with silica, zirconium, and heavily cross-linked organic polymer backbones The polymer columns show the same pressure and solvent fragility described for ion exchange columns Silica size columns must be protected from pH changes like partition columns, which must be used with a pH between 2.5 and 7.5 Zirconium columns are not pH

or temperature sensitive, but possess chelation properties that must be chem-ically masked to prevent interference with the size separation

Much less commonly used than partition, ion exchange, and size columns, affinity columns are of growing interest in the HPLC purification of proteins

AFFINITY CHROMATOGRAPHY 59

because of their very high specificity A molecule with a target site or recognizer is bound to the surface of the affinity packing, sometimes through

a 6-carbon spacer This forms a tight complex with one, and usually only one site, on the compound to be purified The analogy used in affinity separations

is the idea of the lock and key The target site on the compound to be sepa-rated is the key and the recognizer on the affinity packing is the lock that it

fits When a solution containing the target compound is passed down the affin-ity column, only that material with the key functionalaffin-ity is held up and retained

on the column Everything else comes out in the breakthrough volume The target compound can then be eluted with a change in pH, with high salt con-centration, or eluted with a molecule similar to the recognizer lock function

In practice, affinity column recognition specificity is never as complete as described in theory Usually a range or class of similar compounds can be attracted and retained The recognizer must be bound to the column for each target compound and after that point the column must be dedicated for that separation Usually there is no possibility of removing the recognizer and reusing the column for a different separation

The biggest attraction of this type of column is that often it is able to achieve nearly a total purification of the target from a very complex mixture in a single pass down the column Like the ion-exchange column, this type of separation benefits in preparative mode from broad, short columns with a large surface area Its weakness lies in the difficulty of finding and binding the specific rec-ognizer for our target, and in developing optimum eluting conditions