Sample injection volume

For best results, sample injection volume should not exceed... 

SEC measurements were made using a Waters Alliance 2690 separation module with a 410 differential refractometer. Typical chromatographic conditions were 30°C, a 0.5-ml/min flow rate, and a detector sensitivity at 4 with a sample injection volume of 80 fil, respectively, for a sample concentration of 0.075%. All or a combination of PEO standards at 0.05% concentration each were used to generate a linear first-order polynomial fit for each run throughout this work. Polymer Laboratories Caliber GPC/SEC software version 6.0 was used for all SEC collection, analysis, and molecular weight distribution overlays. 

The modem HPLC system is a very powerful analytical tool that can provide very accurate and precise analytical results. The sample injection volume tends to be a minor source of variation, although fixed-loop detectors must be flushed with many times their volume in sample to attain high precision. Assuming adequate peak resolution, fluorimetric, electrochemical, and UV detectors make it possible to detect impurities to parts per billion and to quantitate impurities to parts per thousand or, in favorable cases, to parts per million. The major sources of error in quantitation are sample collection and preparation. Detector response and details of the choice of chromatographic method may also be sources of error. 

As implied above, the appropriate range of sample injection volume depends on column diameter. As we will see in the next section, column diameters vary from capillary size (0.2 to 0.3 mm) to i/8 and i/4 in. Table 12.2 gives the typical injection volumes suggested for these column diameters. The capillary columns are those in which the overloading problem mentioned above is most relevant. Injectors preceding the 1/8 in. or larger columns are not split. 

Assay of the reaction mixture. A 50 /iL sample was removed from the reaction and the dichloromethane component was evaporated under nitrogen for 20 s. The sample was then resuspended in 600 /iL isopropanol and assayed by chiral high-performance liquid chromatography. A 250 mm x 4.6 mm Chiralpak AD-H column was used with an eluant of 85 15 heptane/ethanol, a flow rate of 3 mL min a temperature of 10 °C, a detection wavelength of 245 nm and a sample injection volume of 2 fL. 

Non-Aqueous SEC Experiments. Non-aqueous SEC separations were carried out at ambient temperatures using two Varian MicroPak TSK GMH6 columns in series (7.5mm i.d. x 30cm each). This column is a mixed bed column containing pore sizes from 250 8 to 107 blended to ensure linearity of the molecular weight calibration curve. The mobile phase employed tetrahydrofuran at a flow rate of 1 ml/mln. Sample injection volumes were 50p)l using a Rheodyne 7126 manual loop Injector. 

A gSe of two Waters ultrastyragel columns, designated 10 A and 10 A and a Waters pump (Model 590) for HPLC were used in this study. The elution solvent was tetrahydrofuran (THE) which was distilled in the presence of a small amount of CaH in order to remove the peroxide. The flow rate was maintained at 1 ml/min. The sample injection volume was -30 pi. The chromatogram detected by the differential refractometer (Waters R401) was recorded on a strip chart recorder. All experiments were performed at room temperatures with concentrations below the over-loading condition. 

The detection limit for a 100 yL sample injection volume at 30 ymhos full scale sensitivity is estimated at 0.5 yg/mL. Concentration of samples by freeze-drying affords better detection limits with minimal loss of formic acid. Acetic and carbonic acids are also analyzable under these conditions. 

What happens when the sample injection volume constraint is removed Calculations can be made from the data presented by Cooke et al. (25). For these calculations, the data required is that relating to the maximum sample volumes that can be injected on the various columns while a given number of theoretical plates is maintained. This data, shown in Table IV, was calculated by using eq 3 ... 

By assuming that a proportional increase in the amount of sample injected results in a proportional increase in the detector response for the solute band of interest, the detector response for chromatogram I in Figure 7 will increase 14 times when the maximum sample volume of 7 /xL is injected. However, for the 4.6-mm i.d. column, the detector response will increase 400 times when the maximum sample volume of 200 (lL is injected. By taking into account the relative detector responses for the 0.5-/xL injection, at the maximum sample injection volumes, the 4.6-mm i.d. column with the 20-/liL detector flow cell will produce approximately five times the detector response of the 1-mm i.d. column with the 5-/zL flow cell. In most cases, studies can be designed to provide excess sample because aqueous environmental samples are seldom limited with respect to volume. 

If the injection solvent is of comparable solvent strength to that of the mobile phase, up to 30 ml may be used as the sample injection volume however, if the injection solvent is more lipophilic than the mobile phase, the injection volume is limited to 10 ml. 

Like gated EK injection, gated FID injection was achieved (see Figure 4.23). Sample injection volumes from 0.5 to 10 nL were made in real-time [569]. FID injection conducted in the pinched and gated modes was achieved on a PET chip using three syringe pumps and an eight-port rotary valve [570]. 

Fig. 9 Optimization of sample injection volume. Various injection volumes (VL) of spiked cell lysate samples were investigated to optimize VL. Lysates were spiked with 250 pg/mL of paclitaxel and extracted by selective SPE. The VL was (a) 0.1 pL (b) 0.5 pL (c) 2.0 pL (d) 8.0 pL. A 15 cm x 0.5 mm I.D. capillary column was used for separation the manufacturer-recommended Vnj for the column was 0.1-0.2 pL (Reproduced with permission from Elsevier)...

The typical size of the sample (injection volume) and the concentrations of the components to be analyzed. 

Quantitation of MG Determine the calibration curve and response factor (RF) for Mono-C15 and Mono-C18 by the following procedure Analyze each of the Standard Solutions (Group 1) using a sample injection volume of 0.5 iL. From each chromatogram, establish the response factors (RFi) for the two MG Standards using the following equation ... 

After trimethylsilylation, GC analysis is conducted using a low-polarity capillary column and a flame ionization detector (Note 7). The recommended temperature program is 4 min at 100 °C, then 4°C/min to 240 °C, with the temperature of both injector port and detector at 260 °C. Programs of 2 min at 100 °C and subsequently 5 or 10°C/min to 240 °C are used for GC-MS analysis. Sample injection volumes are 0.2-2.0/d. 

Figure 9.86 Elutions of a stock solution of ATP, ADP, nicotinate, and N MN through a /xBondapak Qs column using 25 mM (NH4)P04 at pH of 8.0. Stock solutions of each reactant were used to assign the peaks. Elution conditions 5 /xL sample injection volumes, 0.7 mL/min flow rate, 25°C. (From Hanna and Sloan, 1980.)...

The 21 NPAHs were determined chemilumigenically with linear calibration graphs from 3 fmol to 20 pmol r > 0.899). The relative standard deviations (n = 3) were less than 5%. The detection limits (S/N = 3) were 1 fmol for the DNPs, 10 fmol for 1-NP, 7-NBaA and 2-NA, 2 fmol for 3-NPer and 6-NBaP, 4 fmol for 9-NA and 1-NPer, 21 fmol for 3-NFR, 30 fmol for 4-NP, 100 fmol for 5-NAc and 4-NPh, 120 fmol for 9-NPh, 150 fmol for 2-NP and 6-NC, 400 finol for 3-NBA, 450 fmol for 2-NTP, 1 pmol for 2-NF, 5.5 pmol for 10-NBA, when the sample injection volume was 100 pL. 

An often-cited advantage of microcolumn LC is that of enhanced detection. However, careful examination of these claims reveals that most often comparisons are made between micro- and traditional LC columns with a fixed sample injection volume. Here there will certainly be enhanced sensitivity with the small-diameter column, as sample dilution is proportional to the square of the inside diameter of the column. However, if the injection volumes onto different columns of different diameters are scaled proportionally to the square of their diameters, then the dilution of the two samples will be equivalent (29). This means that microcolumn LC will only offer enhanced detection sensitivity when the available sample volume is limited. A critical comparison of micro- and standard LC columns in terms of sample detectability using UV absorbance detectors has been made by Cooke et al. (30). 

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