Introduction to Modern Liquid Chromatography, Third Edition part 17 pdf

Autosamplers are best used in a constant-volume injection mode for each method, so the same exact though not necessarily accurate sample volume is injected for both calibration standards

80

60

40

20

0

(a)

(b)

20 μL loop

20 μL

μL dispensed

tube wall

sample

Figure3.19 Effect of laminar flow on injection accuracy (a) Comparison of detector response

when loading different volumes into a 20μL loop; (b) laminar flow profile of sample Adapted

from [12]

simplified description is further complicated by back-flushing and by mixing that takes place when changes in tube diameter or other flow disruptions are encountered

≥200% of the loop volume for maximum accuracy

3.6.2 Autosampler Designs

Autosamplers have largely replaced manual injectors—primarily for convenience, but autosamplers also provide levels of precision that may not be possible with

this level because of errors in calibration of the sample syringe or injection loop Incomplete loop filling due to laminar flow (Section 3.6.1.2) can also introduce volumetric errors Usually the precision of injection is more important than accuracy because of the compensating use of standards or calibrators Autosamplers are best used in a constant-volume injection mode for each method, so the same exact (though not necessarily accurate) sample volume is injected for both calibration standards and samples When this practice is followed, injection accuracy is less important, and the excellent precision will provide satisfactory analytical results Carryover is indicated by the presence of a small peak(s) in a blank chro-matogram (no sample injection) that follows a separation where a sample was injected Carryover results from part of a sample being retained in the system,

mobile phase.

Samples generally are placed in individual sample vials or well-plates containing

96 or 384 sample wells Vials most commonly are made of glass, sometimes specially treated to reduce adsorptive losses of sample Sizes are 1 to 1.5-mL capacity for standard vials and 100 to 300μL (or less) for microvials or inserts in standard vials Vial closures use a cap and septum, usually made of silicon rubber and/or Teflon

closures are usually a press-on septum mat or iron-on metalized polymer film

Sample access most commonly is via movement of the sample needle in xyz

axies to the sample container Some autosamplers use a rotating tray to bring the sample vials to the needle, while others pick up an individual vial and move it to the needle

The cycle time is the amount of time it takes an autosampler to complete

an injection from the time it is given the initial start signal At a minimum, the cycle time includes the time it takes to pick up a sample and inject it onto the column The addition of wash steps or other procedures can increase the cycle time As long as the autosampler cycle time does not have a significant impact on sample throughput, it is not important Thus a 1-min cycle time for a 30-minute gradient run is of little consequence, but the same cycle time would greatly reduce

sec with acceptable levels of carryover and injection precision One way of reducing the negative impact on the autosampler cycle time is to use a ‘‘load-ahead’’ feature offered by some systems In this implementation, the autosampler is programmed to perform its wash cycle(s) and pick up the sample while the previous sample is being eluted As soon as the run is completed, the injection can be made, this reduces the effective cycle time to just a few seconds

Three common autosampler designs are in common use:

• pull-to-fill

• push-to-fill

• needle-in-loop

The pull-to-fill autosampler design is illustrated in Figure 3.20 A syringe is mounted

on a mechanical drive and connected to the injection valve as shown A sample loop corresponding to the desired injection volume is mounted on the valve The needle is mounted on a piece of connecting tubing attached to the valve The needle may be moved to the sample vial, or the vial may be moved to a stationary needle

In the load position (Fig 3.20a) the needle penetrates the septum on the vial and

the syringe plunger is withdrawn to pull sample through the needle and connecting

0

1

c p

(a)

010

0 1

c p (b)

Figure3.20 Pull-to-fill autosampler design (a) Transfer of sample from sample vial to injec-tor loop; (b) injection p, flow from pump; c, flow to column.

tubing until excess sample exits the sample loop The valve rotor then is moved to

the inject position (Fig 3.20b), and the sample is pumped onto the column Note

that no needle seal is used in this type of autosampler

The pull-to-fill autosampler wastes sample because of the relatively large diameter of connecting tubing required to avoid blockage, and the need to flush excess sample through the loop (Section 3.6.1.1) It is therefore best used when the amount of sample is not limited, such as applications used to monitor production processes The design is simple and reliable, and because it uses an overfilled, fixed-volume loop, it can have very good precision and accuracy

The push-to-fill autosampler is an automated version of the manual injector In

the load mode (Fig 3.21a) the needle draws sample from the sample vial into a

connecting tube attached to a mechanically operated syringe The needle then is withdrawn from the sample vial and pushed into the low-pressure needle-seal in the

injection port (Fig 3.21b); sample is then dispensed into the sample loop Next the valve rotor is moved to the inject position (Fig 3.21c), and the sample is pumped

onto the column

The push-to-fill autosampler can be used in the filled-loop or partially filled loop injection mode (Sections 3.6.1.1 and 3.6.1.2) In the partially filled mode, the precision depends on the precision of the syringe controller Because it is possible

to inject nearly all of the sample, the push-to-fill autosampler does not waste much

The push-to-fill autosampler design uses a low-pressure needle-seal that generally is trouble free These autosamplers are very popular and are used for a wide range of applications

0

1

010

0 1

010

0 1

c p c

p

low-pressure needle-seal

Figure3.21 Push-to-fill autosampler design (a) Transfer of sample from sample vial to injec-tion needle and syringe; (b) filling of sample loop; (c) injecinjec-tion p, flow from pump; c, flow to column; w, to waste.

The needle-in-loop autosampler uses a needle and loop that are one piece (needle-loop

in Fig 3.22a) In the load position, as shown in Figure 3.22a, the needle picks up

the sample from the vial The needle is then moved to a high-pressure needle-seal in

the injection port, and the rotor is turned to inject the sample (Fig 3.22b).

Because the tip of the needle is in the flow stream, the needle-in-loop autosam-pler injects all of the sample that is withdrawn from the sample vial, so there

is no wasted sample with this injection technique This makes the needle-in-loop autosampler a favorite for methods in which the sample volume is very small These autosamplers typically use a 100-μL sample needle-loop, which will accommodate most analytical requirements If a larger injection volume is needed, the needle-loop must be replaced with a larger one; this type of needle-loop is much more expensive than a conventional sample loop This autosampler also depends on a high-pressure seal between the sample needle and the injection valve, which is a weak point with some implementations of this design These autosamplers are a popular design because of the low sample waste and generally minimal carryover

3.6.3 Sample-Size Effects

The amount of sample that is injected can influence the appearance of the chro-matogram, not only in peak height or area but also in retention time and peak

and the injection-solvent strength relative to the mobile phase, as discussed below Sample-mass effects and overload are discussed in Section 2.6.2

0 1

c p

(a)

010

0 1

c p (b)

high pressure needle-seal

needle-loop

w w

Figure3.22 Needle-in-loop autosampler design (a) Transfer of sample from sample vial to injector needle-loop; (b) injection p, flow from pump; c, flow to column; w, to waste.

The influence of the injection volume on the peak width was discussed in Section 2.6.1, and is summarized in Table 3.3 and Equation (2.27) as

V p=



4 3



V s2+ V p02

1/2

(2.27)

phase is used as the injection solvent for isocratic separations Equation (2.27) can

be used to determine how large an injection volume can be made for a given increase

in peak volume; if a 5% loss in resolution is acceptable, a 5% increase in peak width can be tolerated The allowed injection volumes listed in Table 3.3 are calculated for a 5% increase in peak width for various columns and retention factors (Note that the allowed injection volumes in Table 3.3 are smaller than those calculated

the sample loop; see Section 2.6.1.) It is obvious that smaller injection volumes are required for smaller volume columns, columns that generate larger plate numbers, and/or early-eluted peaks—all of which result in narrower peaks On the other hand, 4.6-mm i.d columns generate fairly broad peaks, even when packed with sub-2-μm particles and used in short, 50-mm lengths For example, a 50 × 4.6-mm,

1.8-μm column gives V p0 ≈30 μL for k = 0.5, with an allowable sample volume of

Some autosamplers are able to maintain a similar precision for sample

≤3-μm particles, the autosampler must be capable of precisely injecting very small sample

L (mm) d c(mm) d p( μm) N (h≈ 3) k = 0.5 k= 2 k= 20

150 4.6 5.0 10,000 90b(10)c 180 (20) 1,270 (130)

100 1.0 1.8 18,500 2 (<1) 5 (<1) 30 (3)

a Equation (2.27), V s /V p0 = 0.16; note that the latter (theoretical) value of the injection volume V shas been divided by 1.5, to take into account the spreading of the sample plug as it leaves the loop.

bPeak volume ( μL)

cInjection volume ( μL)

volumes In all cases for isocratic separation, the peak width increases with the retention time, so longer retained peaks can tolerate larger injection volumes Modification of a method so as to increase retention is one (seldom used) approach for minimizing extra-column peak broadening, because the resulting reduction in peak height and increase in run time is usually a poor trade-off

As mentioned in Section 2.6.1, when the injection solvent is not matched to the mobile phase, Equation (2.27) no longer holds If the injection solvent is sufficiently weaker than the mobile phase, the sample will be concentrated at the head of the column

Based on a change in k of about 2.5-fold for a change in the mobile phase of 10% B

(Section 6.2.1), an injection solvent 10% B weaker than the mobile phase should sig-nificantly retard the sample as it enters the column; larger solvent-strength differences will be even more effective When large-volume sample injections are desired, dilution

of the sample with water may allow the injection of a larger sample weight (Section 2.6.1) The use of an injection solvent stronger than the mobile phase will adversely affect early-eluted peaks more than more strongly retained ones (Section 17.4.5.3) Also it is important to match the injection solvent for standards and samples Injection in solvents stronger than the mobile phase tends to ‘‘wash’’ the sample down the column until it becomes fully diluted in the mobile phase As with the use of dilute injection solvents, the observed effects are a function of both the injection volume and the difference in solvent strength between the injection solvent

in a strong solvent (e.g., 100% B) generally can be tolerated When larger injection volumes and/or smaller volume columns are used, it is wise to compare retention and peak shape with a small-volume injection of the sample dissolved in mobile

diluting a sample dissolved in 100% B ratio 1:1 with water or buffer will often allow

a larger volume and weight of injected sample without adverse consequences

3.6.4 Other Valve Applications

Automated injection valves are most widely used in autosamplers, but the same valves also are used for other applications These include:

• column switching

• fraction collection

• waste diversion

High-pressure switching valves are available in many configurations other than the simple six-port valve illustrated in Figure 3.17 These may be purchased as motorized valves with switching controlled through the external-events outputs of the HPLC system controller Two general configurations are popular: two-position valves, such

as shown in Figure 3.17, and multi-position valves, which allow a single input tube

to be connected to one of many output tubes (see later the discussion of Fig 3.25) Three applications are discussed here, and another in Section 2.7.6; many additional applications are available on the valve manufacturer’s websites (e.g., [6, 13])

One popular application of the two-position valve, sample enrichment, is

shown in Figure 3.23 The objective is to concentrate a dilute sample and then inject the concentrated fraction onto the analytical column An example of this is the concentration of a nonpolar analyte from a water sample (e.g., an environmental monitoring application) In the enrichment phase the valve is set as shown in Figure 3.23a, where the first pump pushes a dilute sample through an enrichment column, while the previous sample is separated on the analytical column, using a

column in a weak mobile phase to trap the nonpolar materials Once the entire

sample is concentrated on the enrichment column, the valve is switched (Fig 3.23b)

and the sample is back-flushed onto the analytical column Because of the reversed direction of flow and the sudden increase in mobile-phase strength, the sample is released from the enrichment column onto the analytical column in a narrow band for analysis Other applications of column switching as in Figure 3.23 are discussed

in Section 16.9

Figure 3.24 shows a valving configuration that allows regeneration of one

column while a second column is eluting the sample to the detector This can increase throughput and be advantageous for gradient applications, while at the same time increasing the utilization rate of an expensive mass spectrometric detector, such as for the analysis of drugs in plasma samples In the configuration

shown in Figure 3.24a, the sample is injected and analyzed on column 2 in the

normal manner using gradient elution Meanwhile column 1 is regenerated by the mobile phase delivered by a second pumping system As soon as the sample is

Figure3.23 Column switching for sample enrichment (a) Valve set for loading sample enrichment column; (b) back-flushing enrichment column to analytical column.

eluted from column 2 to the detector, the valves are switched to the configuration

of Figure 3.24b, and a new sample is injected onto column 1 while column 2 is

regenerated This application increases throughput by the elimination of the time normally spent waiting for the column to be re-equilibrated to the starting conditions after a gradient run However, it should be noted that other means exist to minimize the time required for column equilibration after gradient elution (Section 9.3.7)

In Figure 3.25, a multi-position valve is used to select from one of three

columns With this setup, three separate columns can be evaluated automatically, for use in method development One application of this technique involves a setup similar to Figure 3.25, but with as many as 32 different chiral columns installed

on two 32-port valves (see Section 14.6.1) In a Gatling-gun approach, a sample

is sequentially injected on each column in an unattended series of runs The chromatograms are later inspected to determine which column provided the best separation

Preparative chromatography (Section 15.2.4) requires fraction collection for either (1) the retrieval of individual peaks from a chromatogram or (2) the collection of fractions from an overlapped peak (as part of the purification of some compound)

A fraction collector, which is commonly used for this purpose, resembles an autosampler that is used in a reversed mode A single sample stream from the column is distributed into multiple vials through use of a mechanism that moves the outlet tube to the desired vial In the simplest implementation a fraction collector is operated on a timed-collection basis At some selected time after injection, collection starts and the sample is collected for a fixed time in each collection tube For

Figure3.24 Column switching for column regeneration (a) Sample is injected on column 2 and directed to detector (MS) while column 1 is regenerated by pump 1; (b) sample is separated

on column 1 while column 2 is regenerated V1, V2, six-port switching valves

Figure3.25 Use of multi-port switching valves for selection of one of several columns As shown valve, V1, directs flow from pump, to column-1, through valve V2, to detector Con-necting passage (- - -) moves to desired column under software control

example, if the peak of interest was eluted at 7 minutes, the fraction collector might

be programmed to start collecting 20-second fractions starting at 6 minutes and ending at 8 minutes This way several ‘‘cuts’’ across the peak would be collected Another popular implementation of the fraction collector is to use an electronic circuit to monitor the detector output so that fractions are collected only when a peak is eluted A delay coil of tubing can be mounted between the detector and the fraction collector to allow for peak detection just prior to the capture of a fraction

The possible applications of switching valves in HPLC are practically limitless Two additional uses of switching valves are discussed here: the diversion of a waste stream

to protect the detector, and the recycle of mobile phase for isocratic applications One method of sample cleanup for the analysis of drugs in plasma is plasma precipitation (Table 16.11) Although quick and inexpensive, plasma precipitation

nonvolatile materials in the injected sample.

Although the cost of mobile-phase solvents is not a large fraction of the total expense of sample analysis, it can be significant when the costs of disposal are considered To reduce this expense, as well as for environmental reasons, some users attempt to reduce the volume of solvents used One approach is to reuse the mobile phase The simplest procedure is to direct the waste stream back to the reservoir (mounted on a stir-plate) As the waste stream is mixed with the remaining mobile phase, impurities are diluted and pumped back into the column at a steady state, so no interfering peaks will appear Over time, however, the contaminant concentration in the reservoir will increase A simple way to minimize this is to recycle only the portion of mobile phase that does not contain sample peaks There are several commercial units (e.g., Axxiom’s SolventTrak) that include a switching valve and a sensor that monitors the detector output When a peak is detected, the valve switches the detector effluent to waste; when no peaks are present, the valve directs the effluent to the mobile-phase reservoir Thus only the ‘‘clean’’ mobile phase is recycled A quantitative evaluation of mobile-phase recycling can be found

in [14]

An alternative way to reduce solvent consumption is to decrease the column internal diameter Solvent consumption is proportional to the cross-sectional area

of the column, so replacement of a 4.6-mm i.d column by a 2.1-mm i.d column will reduce solvent usage by (4.6)2/(2.1)2≈ 5-fold (the flow rate should be simul-taneously reduced by the same amount to maintain a constant linear velocity) The greater importance of extra-column peak broadening for small-diameter columns should be kept in mind, however

It has long been known that column temperature plays an important role in HPLC retention and selectivity For additional information, see Section 6.3.3 and [15–17] (and associated references)

3.7.1 Temperature-Control Requirements

in column temperature will decrease values of k by about 2% Temperature can

also affect chromatographic selectivity (Sections 6.3.3, 7.3.2.2, 8.33, [15]), so close control of column temperature can be important—especially for separations with

preheated to the temperature of the column, distorted peaks can result To avoid peak distortion, the temperature of the mobile phase as it enters the column should