HPLC A Praactical User''''S Guide Part 8 ppt

True preparative is usually run, however, on an HPLC system opti-mized for preparative runs with a much higher flow rate.. If we have to increase load higher, say to obtain a 50-mg sampl

Commercial mobile phase recyclers use peak detection from either a com-puter or an integrator to signal baseline acquisition A waste/recycle valve is triggered by this signal to discard peak containing mobile phase and recycle the baseline There is little solvent recovery for chromatograms containing many closely spaced peaks, but for normal chromatography or in cases where the HPLC is always left running this might represent 40–50% solvent recov-ery Estimates have appeared in the literature that claim that for a quality control instrument run on a single shift this would mean a savings of about

60 L per year at a savings of about $3,000/yr in solvent, man power, and envi-ronmental impact Obviously, the savings would be higher in stat instruments running in clinical laboratories and in quality control laboratories that operate

on a three-shift schedule

134 TROUBLESHOOTING AND OPTIMIZATION

III HPLC UTILIZATION

PREPARATIVE CHROMATOGRAPHY

137

The preparative use of HPLC is often overlooked in the rush to analyze There are two outputs The detector’s electrical signal to the strip chart or computer and the liquid output normally sent to waste Most columns and detectors are nondestructive; aqueous samples can be run without derivatization The same system used for analysis can be used for all but the largest preparative runs Usually, all that is required is a change of columns and a slight modification

of the running conditions to accommodate the increased sample concentra-tion and load (Table 11.1)

Speed, load, and resolution are the three trade-off considerations that must

be balanced to optimize the three levels of preparative runs (Fig 11.1) Ana-lytical preparative is concerned with isolation of up to microgram quantities

of material and with obtaining enough material for spectrometric analysis; the most important factors are speed and resolution

As we move to semipreparative separation, in the milligram range, we are usually purifying analytical standards of recovering impurities to do trace com-pound analysis Resolution is still very important, but now load, not speed, is the trade-off “True” preparative at the gram level can be run using a semi-preparative column in an analytical system by making multiple sequential injections and collecting and combining similar fractions from each chro-matogram True preparative is usually run, however, on an HPLC system opti-mized for preparative runs with a much higher flow rate Load is the major tradeoff; speed is secondary, with resolution the least important Here we are gathering grams of material for biological testing and structural analysis, and

as reaction intermediates

HPLC: A Practical User’s Guide, Second Edition, by Marvin C McMaster

Copyright © 2007 by John Wiley & Sons, Inc.

11.1 ANALYTICAL PREPARATIVE

These separations are run at 1.0–2.0 mL/min on the same 4.5-mm × 25-cm column used for our analytical runs Normally, in analysis we shoot from picogram to nanogram quantities Most separations maintain their resolution until we reach an injection quantity of about 1 mg The valleys between peaks begin to rise indicating some overlapping

If we increase our first peak k′to 8–10, we can increase the interpeak gap allowing us to load to about 10 mg of compound/injection As we increase the amount of sample, we need to go to lower detector sensitivity We can increase flow rate to 2.0 mL/min, but we will lose some resolution by doing so Gener-ally, we have no problem increasing sample concentration and keeping the same injection loop size If necessary, we can increase to the next size larger loop without affecting resolution

138 PREPARATIVE CHROMATOGRAPHY

Table 11.1 A Guide to Preparative Scale-up

Figure 11.1 Speed–load–resolution preparative decision triangle.

If we have to increase load higher, say to obtain a 50-mg sample for NMR analysis, we can use the shave/recycle technique to be described in the “true” preparative section These runs must be made isocratic and column overload occurs at 100–150 mg for most compounds If this much material is needed, it

is better to switch to a semipreparative column, which can easily produce mil-ligram quantities in a single pass

Semipreparative separations are made on a 10-mm × 25-cm column packed with the same 5- or 10-mm packing used in the analytical separation Simply replace the column and equilibrate with the analytical mobile phase used in analysis A 1–5-mg sample can be injected with a flow rate, FR2, calculated from the following formula:

FR2=FR1×(D2/D1)2 Where FR1is the analytical flow rate, D2is the semipreparative column diam-eter, and D1is the diameter of the equivalent analytical column and we use these to calculate the square of the column diameters differences With our 10-cm column we would use a flow rate of 5 mL/min

By using solvent polarity techniques to increase k′we can push the load to

20 mg Going isocratic and using shave/recycle, the load can be increased to

100 mg with column overload occurring at 200–300 mg injections

11.3 “TRUE” PREPARATIVE

Preparative separations in the grams per injection level are different Separa-tions are run isocratic in 1- to 3-in columns with large pore, fully porous pack-ings (35–60 mm) An analytical, two-pump system can just barely reach the 20-mL/min flow rates needed to run a 1-in column Special preparative HPLC systems deliver flow rates of 50–500 mL/min to handle the larger bore columns

A stream splitter is used to send part of the flow through a refractive index detector with a flow cell designed for concentrated solutions

Injection samples need to be as concentrated as possible and this leads to problems A column acts as a sample concentrator If the solution starts out saturated, it will supersaturate on the column, precipitate, and plug the column I have seen a column with a 3-cm-deep plug that had to be bored out with a drill bit and a spatula A couple of injector loops full of the stronger solvent in a mixed mobile phase will clear this if there is still some flow, but the separation will have to be repeated It is better to dissolve the compound, then add a half volume of additional solvent, ensuring that there will be no precipitation on injection

“TRUE” PREPARATIVE 139

A technique called shave/recycle, mentioned earlier, allows separtion of a

pair of close resolving peaks To use shave/recycle, it is necessary to plumb the HPLC system so that the output from the detector can be returned to the HPLC pumps through small diameter tubing and switching valves (Fig 11.2) Twenty-thousandths tubing is used to connect the detector output to valve 2, the waste recycle valve; 0.02-in tubing connects from valve 2 to valve 1, the solvent select valve; and, finally, a third valve 3, the collect valve, can be placed

in the waste line from valve 2

The analytical separation is used as a guide to selecting load conditions for

the preparative run First, k′(Fig 11.3a) is increased until the first peak of the

desired pair of peaks to be separated comes off with a k′ = 10 (Fig 11.3 b) Load is scaled up until the valley between the two peaks is just visible (Fig 11.3c) If there are peaks running later than our target pair, we will have to inject the sample and collect the fraction containing the compounds of inter-est for reinjection If the only impurities come off before the target pair, the impurities can be discarded after making the shave/recycle injection The preparative instrument can be a little intimidating to run because things happen so fast at maximum flow rate With a top flow rate of 500 mL/min, a liter flask is filled in 2 min The first time you run the analysis, I would suggest using the slowest flow rate possible to acclimate yourself

To begin a run (Fig 11.3d), a sample must first be injected with valve 1 turned to the reservoir and valves 2 and 3 to waste You can use either a very large loop and valve injector or a stop flow injection in which the sample solu-tion is pushed through the solvent line through an injecsolu-tion port, or you can pump the sample in using either the main HPLC pump or an analytical, loading pump plumbed in through a three-way valve 1 After injection (Fig 11.3d), you will see the void volume peak followed by any early running impu-rities, which are all run to waste If you are using a loop and valve injector,

140 PREPARATIVE CHROMATOGRAPHY

Figure 11.2 Recycle system.

wash through the loop with six-loop volumes of mobile phase, then turn the

handle back into the load position.This is important because it removes a major

source of dead volume from the recycle pathway

As your first peak begins to elute, switch to collection of fraction 1 by turning valve 3 to collect Continue to collect until you are over the maxima

of peak A and one-third of the way down to the valley between the peaks (Peak A tails badly into peak B, but there is little of B in peak A until we get well into the rear slope of A Be brave, you can always reinject if your ana-lytical system shows you were too late in your cut.)

At this point, switch to recycle by turning valve 1 and 2 at the same time; switch valve 3 to waste We want to send the contaminated portion between peaks back through the pump head back to the column head for further sep-aration We continue to recycle until the detector shows we are well down on the backside of peak B (Remember, A is tailing into B.) Change the collec-tion flask while we are recycling to collect flask 2 Shut recycle valves 1 and 2 and switch valve 3 to collect in flask 2 Stop collecting 2 when we reach the baseline on the recorder and switch valve 3 back to waste and change to a clean collection flask 3

Very quickly, peak A should begin emerging from its second pass through the column Switch valve 3 and begin collecting faction 3 in its clean flask We

“TRUE” PREPARATIVE 141

Figure 11.3 Shave recycle (a) Analytical; (b) k′ increase; (c) overload; (d) preparative shave/recycle.

will continue the cycle: 1) collect peak A, 2) recycle the middle by opening valves 1 and 2 and switching valve 3 to waste, and 3) close valves 1 and 2 and switch valve 3 from waste to collect the next fraction of B in a clean flask, until

we have separated the peaks (three passes is usually enough to reach base-line) The slow flow rate is fine for the beginners, but you will find yourself quickly using only the maximum flow rate of the systems Have plenty of vol-unteers on hand People will be rushing around with flasks of collected sample trying to get to a flash evaporator before sample starts to crystallize

Once all fractions are collected, we can take them to our analytical appa-ratus to ensure purity, then combine odd fractions—1,3,5, and so on—for recovery for peak A, and even fractions—2,4,6—for recovery of peak B Volatile, organic solvents can be rotary evaporated for sample and solvent recovery

Evaporation of large volumes of water mixture from samples eluted from reverse-phase columns can be very time consuming Instead, you can use the preparative HPLC to recover pure compounds from aqueous solution Dilute the combined fractions from peak A 5- to 10-fold with water and pump them back onto the column, either through the injector or through the pump

Dilu-tion increases the compound’s k′, causing it to be retained strongly at the column head Then, immediately elute and collect the compound with a strong solvent like methanol Your sample can now be recovered rapidly by rotary evaporation from the relatively small volume of strong solvent needed for this elution Each purified compound can be recovered in turn using the same technique

A commercial customer had four compounds to recover from a synthesis mixture; they separated as two pairs of compounds They injected and col-lected each pair together, then diluted each pair with water, reinjected onto the reverse-phase column, and ran shave/recycle Using this technique they purified 50 gm of each compound in the two injections of the pair fractions on the 3-in column

142 PREPARATIVE CHROMATOGRAPHY

SAMPLE PREPARATION

AND METHODS DEVELOPMENT

143

Sample preparation is the key to getting the most out of an HPLC system Unless you are working with purified standards or examining a compound in

a very pure matrix, your chromatography becomes complicated with extrane-ous compounds

A biological matrix such as serum provides a good example Nature gener-ally tends to make metabolites more polar than the original compound These polars exist to aid in elimination and excretion as well as serving as building blocks and reaction components At the same time, nonpolar molecules are present in transport and structural roles and end up in the circulating blood The effect on chromatography is to complicate the separation greatly If we consider a reverse-phase separation, the first thing we notice is an almost irre-versible binding of protein to the column Even after protein removal, we find polar peaks, which overload the early part of the chromatogram and tail into the compounds of interest The components that are more nonpolar than our compounds of interest adhere to the column and must be washed off before the next injection To ensure polar elution before our target compounds and nonpolar removal afterwards, we are almost forced to run solvent gradients Sample preparation techniques are aimed at removal of as many of these extraneous materials as possible before injection onto the column The expected result should be a dramatic reduction in run times, hopefully to a fast running isocratic separation instead of a gradient A side benefit of much of this sample preparation is often trace enrichment, an increase in sample con-centration with a corresponding increase in detectability

HPLC: A Practical User’s Guide, Second Edition, by Marvin C McMaster

Copyright © 2007 by John Wiley & Sons, Inc.

The first step in preparing a sample for injection is to ensure it is completely dissolved and to remove particulate matter If we are working with blood samples, we need to get rid of blood cells, which is done by centrifugation Be aware that in removing both particulates and red blood cells, there is a chance that you may remove some of your compound of interest This needs to be checked by adding known amounts of your target compounds to a represen-tative blood sample, removing the interfering materials, and then quantita-tively checking your recovery of added standards

12.1.1 Deproteination

The next general step is to remove charged molecules that interact with silica Since silicic acid is a weak cationic ion exchanger, the compounds we are trying

to remove will be positively charged In serum, the most common of these are proteins, especially nonpolar proteins such as albumin Proteins also can inter-act with bonded-phase columns through nonpolar partitioning It is usually best to avoid putting them on the column since removal is difficult and time consuming

As mentioned earlier, proteins can be removed by ultrafiltration through a very fine membrane filter Ultracentrifugation at high speeds can also be used

to separate proteins from smaller molecules based on size differences The most commonly used protein removal techniques for HPLC involve protein denaturation Heating denatures most proteins If the compounds to be sepa-rated are temperature resistant, the crude mixture remaining can be boiled and then filtered or centrifuged Particulates and denatured protein are removed together

Chemical denaturation of proteins tends to be more efficient and less harmful to sensitive compounds Acidification with trichloracetic acid (TCA; 5% in final solution), centrifugation to remove protein, and neutralization with sodium hydroxide remove better than 99% of the protein A second reagent used for protein precipitation is perchloracetic acid After protein precipita-tion occurs, excess perchloracetic acid is precipitated as KClO4by neutraliza-tion with potassium hydroxide Both of these acid treatments, however, suffer from problems TCA absorbs strongly below 230 nm, eliminating the use of low-wavelength detection The perchloracetic acid treatment leaves large amount of salt in solution, which can precipitate with organic solvents or cause major early refractive index upsets of the UV baseline

Probably the best chemical precipitant for use in HPLC is acetonitrile Ace-tonitrile has the advantages of being a common solvent for HPLC and of being

UV transparent to 190 nm Mixing and centrifugation of an equal volume of plasma sample solution and acetonitrile will lead to precipitation of about 95%

of the proteins; nonpolar proteins, such as the albumins, remain in the liquid phase The supernatant can be injected directly if a guard column is used to remove the last 5% of the protein The guard column will need to be repacked

or inverted and washed out to a beaker periodically to prevent protein break-through to the main column

144 SAMPLE PREPARATION AND METHODS DEVELOPMENT