of sample. The upper limit to sample size is determined largely by the available column end space and by the bore of the column. We have found that 100-wl samples may safely be injected onto a 6.2-mm i.d. preparative column, while a 1-mm i.d. analytical column gave good linearity of response for sample sizes of 1 to 10 wl (the correlation coefficient was 0.9998 for a plot of peak area vs. sample volume), and was still usable with a sample volume of 25 ~1 (corresponding to a 3.2-cm length of 1-mm i.d. column). Peak areas from repeated 1O-wl injections had a standard deviation of about 2%. As an example of the use of this device, Figure 2 shows the result of making six injections each of 2 microliters of a mixture of the two isomers of adenylyladenosine (2’,5’- and 3’,5’-linked) from a standard Hamilton 10-microliter syringe.
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LITERATURE CITED
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Figure 2. Reproducibility of the septumless injection port Six injections each of two microliters were made from a Hamilton ten-microliter syringe. The sample was a mixture of 2’,5’- and 3‘.5’-adenyiyl-adenosine (0.26 and 0.22 mg/ml respectively: Sigma Lot 81C-1790 and Lot 13C-2240); the column was 1-mm X 1-meter AS-Peilionex-SAX (Reeve-Angel, lot 152010): the eluent was 0.01M mmonium sulfate, with a flow rate of 0.2 ml/min. A Waters Associates ALC-202 with model 6000 pump and UV detector was used
typically cost several hundred to over a thousand dollars, although the argument is made that they pay for themselves relatively quickly in the form of reduced outlay for new syringes and septa. The use of a Swagelok union-tee as an injection port has been described by Horvath in a recent review ( I ) , but many users of liquid chromatographs are apparently still unaware of the ease of construction and use of such a device. Our version of a septumless injection port uses an unmodified tee and costs about ten dollars. The injector is shown disassembled in Figure 1, left and assembled in Figure 1, right. The column shown is j/s-inch o.d., and was coupled directly to a standard Swagelok SS200-3 union-tee; the front port being closed during operation by a short length of fi8-inch 316 stainless steel rod. In operation, the flow is stopped and the front rod removed. The sample is then injected gently directly onto the column and the rod replaced. The short projection of the rod into the injector opening displaces any air again and, if in doubt, the operator could always run the pump briefly to fill the injector opening with more liquid, though in practice we have not had to take this precaution. After the rod is replaced and the nut tightened, the flow is restarted. Sample injection with intermittent flow has in general not resulted in inferior separation compared to that obtained with continuous flow. When the internal bore of the column is much smaller than the bore of the union, care must be taken that the sample is injected into the column itself, and not just into the union (radial diffusion is slow, and broad peaks may result if the sample encounters an abrupt change in bore size). A little packing material can be removed from the end of the column to allow space for the needle to deposit the sample and, if necessary, a few plain glass beads can be placed a t the end of the packing to stop the needle from becoming plugged by fine particles of the packing material. Using this system, and a minimum amount of care, it is possible to get reproducible results on less than a microliter
784
ANALYTICAL CHEMISTRY, VOL. 47, N O . 4 , A P R I L 1975
(1) C. Horvath, “Methods of Biochemical Analysis,” D. Glick, Ed.. John Wiley & Sons, Inc., New York, NY, Volume 21, 1973, p 104.
RECEIVEDfor review July 15, 1974. Accepted January 9, 1975. ADDENDUM Comparison of Cell Extraction Procedures for Use with High Pressure Liquid Chromatography
I t has come to my attention that the procedure recommended for extraction of nucleotides in cell extracts in our article ‘Comparison of Cell Extraction Procedures for Use with High Pressure Liquid Chromatography,” published in Analytical Chemistry, 44, 1072 (1972), is not universally applicable with all columns. With the pellicular anion exchange packing which we used for the past five years, the extraction procedure worked well. However, with the newer microparticle packings, the high concentration of tris salts in the solution can cause poor resolution of the nucleotides and problems with the columns. Because of the rapid development of new packings and HPLC technology, it is advisable for researchers to do preliminary work to determine the extraction procedure best suited to the column and operating conditions used for’ their particular analyses. Other extraction procedures are now being investigated and, hopefully, in the near future, improved extraction procedures will be developed which can be used with all columns. Phyllis R. Brown Department of Chemistry University of Rhode Island Kingston, RI 02881 CORRECTION Water-cooled Condenser for Unattended Operations
It has been pointed out that the set-up shown in Figure 1 in this paper by David J. Stanonis, Anal. Chem., 46, 2255 (1974), may produce backflow. This situation can be corrected by the use of a suitable air gap which can be accomplished by removing the connecting hose from the water faucet into the beaker.
