Effect of Temperature Control on the Stability and Sensitivity of a High Pressure Liquid Chromatography Ultraviolet Flow Cell Detector Gary Brooker Departments of Medicine and Biochemistry, University of Southern California, School of Medicine, Los Angeles, Calif. 90033
and the chromatographic effluent stream which passes through the detector cell. The present paper describes the modified detector system and its characteristics and sensitivity in high pressure anion exchange chromatography. The sensitivity afforded by these modifications permits routine quantification of nucleotides in picomole amounts. It appears that the modifications described herein would also be applicable to other micro spectrophotometric flow cell detectors.
HIGH PRESSURE LIQUID CHROMATOGRAPHY is playing a n increasing role in the analysis of many compounds which can be easily separated on liquid chromatography columns. One advantage of liquid chromatography over that of gas chromatography is that derivative formation is not needed for analysis of many nonvolatile compounds. One disadvantage, however, is that the detectors available for liquid chromatography columns d o not a t present have the sensitivity of many gas chromatograph detectors. The detector employed in the Varian LCS 1000 liquid chromatograph appears to be the most sensitive liquid chromatography detector for compounds absorbing a t 254 nm. This detector can detect nanomole and, under certain conditions, picomole amounts of nucleotides and nucleosides eluted from “pellicular’’ anion and cation exchange columns ( I , 2). It is advantageous to make these analyses at high flow rates to make the analysis more rapid. However, high flow rates and pressures used in this system create excessive noise, making it impossible t o measure picomole amounts of these substances. At temperatures of 70-80 “C the exchange rate of the resin is increased sufficiently to make high flow rate anion and cation exchange chromatography feasible. Since a higher noise level in the ultraviolet flow cell detector would occur a t this temperature, it is mounted outside of the column oven and its temperature is not under the precise control imposed upon the column. The stability and sensitivity of this detector can be markedly improved by modification to permit temperature control of the detector
EXPERIMENTAL
Apparatus. A Varian-Aerograph LCS 1000 liquid chromatograph equipped with a n 8-pl, 1-cm path length, 254-nm ultraviolet flow cell was used. The column pump was a 3000-psi Milton Roy “Milroyal” piston pump. The chromatograph was equipped with a 3-meter capillary “pellicular” anion exchange column as originally described by Horvath et al. ( I ) . A Varian-Aerograph potentiometric 1-mV strip chart recorded was used with full scale deflection varying between 0.002 and 0.016 absorbance, A , depending upon the setting o n the chromatograph. Reagents. Adenosine and adenosine 3 ’,5’-cyclic monophosphate were obtained from Sigma Chemical Company, St. Louis. Solutions of known concentration were made in a Beckman DB Spectrophotometer from published absorptivities (3). All other chemicals used in this study were ACS reagent grade or better. Procedure. High pressure anion exchange chromatography was performed as described previously ( 2 ) with HCI p H 2.20 as the eluent. One notable exception was the markedly
__ (1) C. G . Horvath, B. A. Preiss, and S. R. Lipsky, ANAL.CHEM., 39, 1422 (1967). (2) G . Brooker, ibid., 42, 1108 (1970).
(3) M. Smith, G . I. Drummond, and H. G . Khorana, J. Amer. Chem. SOC., 83,698 (1961).
coolant
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Figure 1. Diagrammatic representation of the improved ultraviolet flow cell detector
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Figure 2. High sensitivity base-line stability of the chromatograph without any added time constant A . Before modification B. After the temperature control modifications
increased flow rate of 25 to 57 ml/hr used during this study, with inlet pressures ranging from 1100-1250 to 3000-3100 psi, respectively. During high pressure ion exchange chromatography, the eluting solvent was pumped into the ion exchange column enclosed in a n oven maintained at 80 "C. The effluent from the column left the oven through a short piece of stainless steel capillary tubing and entered the ultraviolet 254-nm detector which was positioned adjacent to the oven, a t ambient temperature. Because of the short distance from the column end t o the detector, there was not enough time for the column effluent to reach the ambient temperature of the detector cell assembly. To obtain the highest sensitivity, it was necessary to operate the chromatograph at very low flow rates on the order of 3-5 ml per hour (4). As the flow rate was increased, the base-line noise from the ultraviolet detector also increased. Thus it became apparent that factors other than inherent electrical noise must be involved in the noisy base line a t higher flow rates. One major factor appears to be the temperature of the column effluent during its flow through the ultraviolet absorption flow cell. To eliminate this temperature differential during passage through the cell, it was modified as shown in the diagram of Figure 1 . Ten feet of 0.020 0.d. X 0.010 i.d. (volume = 150 pl) stainless steel capillary tubing was coiled and encased in a water jacket, The flow cell was also encased in a machined copper jacket which had heat exchanger cooling coils soldered to its body. The sample flow then consisted of exit from the chromatographic column, passage through the water-jacketed cooling coil, and then entrance into the water cooled ultraviolet flow cell. Thus the effluent could be maintained at a constant temperature at any flow rate. The chromatographic effluent thus did not change in temperature while passing through the ultraviolet flow cell. Tap water (T = 20 "C) was run through the cooling coils to maintain the column effluent and the detector at the same temperature. In addition, the optical section of the flow cell was mounted approximately 1 in. above the ultraviolet lamp to allow more heat generated from the ultraviolet source to dissipate. (4) C. G . Horvath and S. R. Lipsky, ANAL.CHEM., 41, 1227 (1969). 1096
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Figure 3. High sensitivity base-line stability of the chromatograph with 0.25second time constant A . Before modification
B. After the temperature control modiflcations
Another factor employed without seriously affecting the chromatographic system was the insertion of a 500,000-ohm resistor in series and a 0.5-pF capacitor in parallel with the output signal from the detector. This gave a time constant of 0.25 second, which effectively filtered out the inherent electrical pulsations of the UV detector a t high sensitivity. The instrument was standardized using solutions of adenosine made up to absorbance 0.30 at 254 nm and diluted appropriately. The solution was pumped directly through the detector and the gain was adjusted in the feedback power supply of the instrument to obtain the proper recorder deflection for each absorbance tested. The detector response was found to be linear. RESULTS
Effect of Temperature Control on Base-Line Stability. For efficient high pressure ion exchange liquid chromatography, it is essential that high sensitivity be coupled to high speed analysis. Figure 2A shows a typical chromatogram from the LCS 1000 at a high sensitivity without any of the above modifications. It can be seen that the short term noise level a t a flow rate of 25 ml/hr is 0.0006 Almin, similar to that guaranteed by the manufacturer. This chromatogram was obtained without the use of any added time constant to the instrument. Figure 2B shows the noise level obtained and drift of the instrument with the temperature control system added. Again, no time constant was applied to the output of the instrument. A significant decrease in noise was observed. Short term noise level with this modification was about 0.0001 Almin. Base-line stability was then evaluated in the presence of the 0.25-second time constant with and without the temperature control modifications. At a flow rate of 57 ml/hr in the absence of the temperature control system, the short term
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0.002 cyclic AMP
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Figure 4. Effect of changing flow rate on base-line stability A . Before modification B. After the temperature control modifica-
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noise was 0.0002 Almin and the long range drift rate was about 0.002 Alhr as seen in Figure 3A. As shown in Figure 3B, the temperature control system markedly improved the base-line stability and sensitivity, even a t flow rates of 57 ml/hr. With the temperature control, short term noise was reduced to less than 0.00004 Almin, and the long range drift rate was less than 0.00004 Alhr. Effect of Changing Flow Rate-on Base-Line Stability. It was virtually impossible to make flow rate adjustments during a chromatographic run with the unmodified equipment, because the base line of the ultraviolet detector was very sensitive t o changes in flow rate. As shown in Figure 4A) the chromatograph was run with chromatographic conditions described in the procedure section using a time constant of 0.25 second. Figure 4A is a typical chromatographic trace of the instrument before the above modifications were made. When the flow was abruptly changed from 25 ml t o 57 ml per hour, there was a n abrupt change in the absorbance greater than 0.008 A unit. A new base line was then subsequently established a t a new and higher base line. To demonstrate the dramatic improvement that temperature control yields in base-line stability with changing flow rates, the sensitivity was increased fourfold in trace 4B. The flow was again changed from 25 ml to 57 ml per hour. No detectable change in base line was observed in the record, even when using four times higher sensitivity than in trace 4A. Figure 5 shows a practical use of the LCS 1000 with the modified ultraviolet detector for the measurement of cyclic A M P in picomole amounts. Flow rate was 25 ml/hr. Ten pl of solution containing 43 picomoles of cyclic A M P was injected into the chromatograph, and the flow was started. The cyclic AMP peak gave almost a full scale recorder deflection when this amount of cyclic A M P was chromatographed. The peak width for chromatography of cyclic AMP was similar before or after the detector was modified.
chromatograph and improved detector for measurement of cyclic AMP. 'The peak represents 43 picomoles of cyclic AMP DISCUSSION
Routine high pressure chromatography of picomole amounts of ultraviolet absorbing compounds requires a sensitive and stable detector. The simple modifications made to the Varian ultraviolet detector described in this paper now for the first time permit picomole measurements a t high flow rates. These modifications appear to have extended the high flow rate stability and sensitivity of the instrument to about that found by Horvath and Lipsky ( 4 ) when the unmodified equipment was examined in the absence of any flow. Typically a t flow rates of 25-50 ml/hr, 50 picomoles of a nucleotide can now be eluted in 1 ml, yielding full scale recorder peaks. If special chromatographic conditions are employed to elute the sample in a n even smaller volume, still higher sensitivity is possible. This improved detector system in combination with the Varian LCS 1000 has now been used to separate and quantitate cyclic A M P in samples of biological materials containing less than 15 picomoles of the nucleotide. The exact details of this method will be the subject of another report. Because this modified detector is not sensitive to flow changes, flow programming can now be considered for high pressure ion exchange liquid chromatography. In addition to changes in column temperature and salt concentration of the buffer, intermittent increases in the flow rate between peaks can now be ysed to further reduce the total analysis time for a complex sample. ACKNOWLEDGMENT It is a pleasure to acknowledge the expert technical assistance of Sharon Laws and the meticulous secretarial work of Georgene Denison.
RECEIVED for review February 24, 1971. Accepted April 21,1971. This work was supported in part by a Grant-InAid from the Los Angeles County Heart Association, the American Heart Association, the Diabetes Association of Southern California, and the University of Southern California. ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
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