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

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 8–12mL; 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

PARTITION 51

Table 4.1 Relationship of efficiency to flow rate

Efficiency Changes with Particle Size

Packing diameter (mm) Plates/meter Flow rate (mL/min)

10 30,000 1.0

5 50,000 1.5

3 100,000 2.5

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; VO used

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

the VO distance (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 things 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.

52 SEPARATION MODELS

Figure 4.5 Retention factor equation.