Position calibration

Establishing a calibration function with one single broad distributed sample is an alternative to traditional peak postion calibration of SEC systems with a set of narrow distributed standards. An obvious advantage of this technique is time for peak position calibration elution profiles for the set of standards need to be determined for broad standard calibration the elution profile of one sample needs to be determined only. Establishing a linear calibration function with a broad distributed standard includes startup information [M (true), Mn(true)] and an iterative (repeat.. . until) algorithm ... 

TABLE 16.7 Molecular Weight and Polydispersity of Standards for Peak Position Calibration of Superose 6 HR 10/30... 

Figure 4, Comparison of broad standard with peak position SEC-[rj] calibration curves ([rj = loge [7/] = loge El — EgV (-0-) peak position calibration...

The peak position and universal calibration methods rely on peak position calibration with known polymers of narrow molecular weight distribution. Several other calibration procedures requiring only a single broad moleculau weight standard have been proposed [77,439]. These procedures are quite c< plex and have a major drawback in that, unlike the peak position methods, instrumental peak broadening must be accounted for correctly if accurate results are to be obtained. 

Geometry +1 Wrong position of sample in X-ray or neutron beam, difference between sample and calibrant position Calibrant and unknowns both in (he same form e.g. solution or powder measured in identical vials at the same distance of the detector check for chamber background... 

To determine the size of unknown silica sols with relatively narrow distributions, the retention time of the band for an unknown sample is compared to a peak-position calibration plot such as that shown in Figure 9. Just as for the determination of molecular weight for polymers, broad distributions of silica sols can be measured with appropriate software by using known calibration methods involving peak positions or by using known standards with broad distributions (26). However, commercial software for specifically characterizing silica sols and other colloids is apparently not yet available. 

Relatively small fractionating volumes are associated with HDC systems. Therefore, the Rf market method (27) typically is used to compensate for possible variations in the operating parameters during the separation. Figure 13 shows a HDC calibration plot that was obtained by the marker method. The arbitrary log sol diameter versus Rf plot produced a linear relationship for this series of 40-600-nm SdFFF-characterized sols as standards. With this particular HDC system, silica sols can be routinely measured with precisions of about 15% (relative) with the peak-position calibration method. This precision level is a direct result of the relatively poor resolution of HDC separations. 

Figure 12.5 Position calibration. Left a bead is held in the optical tweezers and a 1 Hz squarewave oscillation is applied to the AODs the displacements are (a) 50, (b) 100, (c) 200 and (d) 500 nm. Right a large amplitude triangle wave is applied to the AODs to move the bead held in the optical tweezer over the full active area of the detector. The sensitivity is greatest when the bead is in the centre of the 4QD. Ideally the image of the bead held in the optical tweezers should be approximately half the size of the 4QD. This gives greatest sensitivity and suflident detector range to perform experiments...

Since the user will never be totally satisfied with automatic analysis all the time, it is very important to allow him to assume control over all parts of the quantification whenever necessary. For example, the peak analysis routine of FSUNMR performs an automatic quantification by default. The user is given a wide range of options when the computations are complete. These include insertion and deletion of peaks, manual declaration of a threshold, peak position calibration, and a complete set of display options. Good peak analysis routines can save the spectroscopist a lot of time without... 

Figure 4.4.17. Description of the principal construction of a Knauer membrane osmometer A 300 1 - head thermostat, 2 - channel for syringe, 3 - calibration device with suction tube, 4 - calibration glass, 5 - c illary position MEASUREMENT, 6 - capillary position CALIBRATION, 7 - tension screws, 8 - cell retaining disc, 9 - upper half of measuring cell, 10 - sample introduction system, 11 -semipermeable membrane, 12 - lower half of measuring cell, 13 -pressure measuring system, 14 - cell thermostat, 15 - suction of calibration bottle. [Reprinted from the operating manual with permission from Dr. H. Knauer GmbH (Germany)].

Obviously, the simplest way to precisely calibrate the peak areas is to make a separate run with a known sample volume containing elution buffer as the semple ("external calibration"). Likewise, a known excess of ligand can be applied to get a positive calibration peak. The calibration can also be carried out by repeating HD runs with samples of M and varying amounts of L ("internal calibration"). With a certain amount of L, no negative peak will appear (ref. 42,51,62,63). The external and internal calibration have been proven to yield equal results (ref. 59). The external calibration has the advantage of not consuming the sample. 

If you are not using an FT-IR spectrometer, it is good laboratory practice to calibrate the experimentally obtained IR spectra against a standard that has absorption bands at known positions. Calibration is necessary because problems such as misalignment of the chart paper in the instrument or mechanical slippage may result in absorptions appearing at wavenumbers that are incorrect. Polystyrene (Fig. 8.10) is a commonly used standard and is typically available as a thin, transparent film in a cardboard holder that fits into the spectrometer. After the spectrum of the sample is obtained, the standard is inserted in place of the sample cell the reference cell, if used, is removed and at least one band of the standard is recorded on the spectrum of the sample. In the case of polystyrene, the absorption at 1601 cm is typically recorded, although other bands may be used. 

During the position calibration procedure, the displacement transducer used to measure position is calibrated against a height displacement standard (usually made from a hard material such as sapphire) with a precisely known height. Current TMA instruments can measure probe position changes as small as lOnm (see discussion of LVDT in Section 4.3.1). 

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