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(4) Schieffer, Gary W. Anal. Chem. 1980, 52, 1994-1998. (5) Schieffer, Gary W. Anal. Chem. 1985, 57, 968-971. (6) Schieffer, Gary W. J . Chromafogr. 1080, 202,405-412. (7) Behner, E. Dale; Hubbard, Richard W. Clln. Chem. (Winston-Salem, N.C.), 1979, 25, 1512-1513. (8) King, William P.; Kissinger, Peter T. Clin. Chem. ( Winston-Salem,
N.C.), 1080, 26, 1484-1491, (9) Evans, Dennis H., University of Wisconsin-Madison, communication, 1985.
personal
RECEIVED for review June 19, 1985. Accepted July 22, 1985.
Applications of a Computerized Flow Programmer for Capillary Column Gas Chromatography Soren Nygren*’
Department of Analytical Chemistry, University of Uppsala, P.O. Box 531, S-751 21 Uppsala, Sweden Svante Anderson
T h e National Swedish Laboratory for Agricultural Chemistry, S-750 07 Uppsala, Sweden It has been shown (1-4) that exponential flow programming in capillary gas chromatography under isothermal conditions gives peak distributions which are very similar to those obtained with temperature programming. The technique might thus be used as a useful alternative to temperature programming. With flow programming the elution of the higher boiling components will be accelerated by the steadily increasing flow rate of the carrier gas. This leads to more evenly spaced peaks in the chromatogram. Experience shows that peak widths are not adversely affected by the high flow rates at the end of the separation. In fact, the peak widths stay almost constant, but the efficiency is somewhat reduced at the end of a run because the flow rate is not optimal in the last part of the flow program. With mass sensitive detectors the high flow rates are beneficial since the signal to noise ratio increases. Flow programming is particularly well suited for short or wide-bore capillary columns, which permit separations to be achieved in a few minutes (5). Since only the pressure drop over the column needs to be reset before the next run, the sample throughput can be made very high. The technique is, however, not well adapted to packed columns due to the high flow resistance of such columns. The range of boiling points which can be covered in a flow programmed run is relatively large but not as broad as with temperature programming. The two techniques can, however, be combined in order to cover a wide range of boiling points. Since separation can be carried out a t a lower temperature with flow programming than with temperature programming, the former technique is well suited for separations of thermolabile compounds and also reduces bleeding of the stationary phase. No peak order reversals will occur as is sometimes the case with temperature programming. The flow function Q, = Q,
+ Qoekqtr
used for exponential programming gives the relationship between the retention flow rate, Q,, and the retention time, t,. The parameters Q, and Qo together with the programming rate, K,, determine the range and rate of the program. The sum of Q, and Qo is equal to the starting flow rate. The retention volume, VI, obtained by integrating Q, dt between the limits t = 0 and t = t,, is
V , = Q,t,
+ Qo/kq(ekqtr- 1)
(2)
The present paper describes a flexible, microcomputer conPresent address: Pharmacia AB, s-751 82 Uppsala, Sweden. 0003-2700/85/0357-2748$01.50/0
trolled device which, by controlling the gas pressure, regulates the flow of the eluting gas according to the flow function. The effectiveness of this method is demonstrated by reference to a number of typical applications using various conditions and chromatographic equipment.
EXPERIMENTAL SECTION Chromatographic Systems. The chromatographsused were a Pye unicam 204 with a splitless column injection system coupled to a FPD detector, a Pye Unicam GCD equipped with a “falling needle” injector for capillary columns coupled to a FID detector, and a Pye Unicam GCV with a splitless column injection system and an ECD detector (“Ni). The samples separated were a standard mixture of organophosphorus pesticides, two different mixtures of polyaromatic hydrocarbons (PAH), and an extract from Baltic herring containing chlorinated hydrocarbons. The columns used were AmAc, 25 m, 0.40 mm i.d., SE-54, 50 m, 0.30 mm i.d., and SE-30, 25 m, 0.30 mm i.d. Flow Programmer. A general view of the flow programmer is shown in Figure 1. The personal computer was an ABC 80, Luxor, Sweden. The flow rate of the eluting gas can be regulated in two ways. The first method uses the calibration curve of the gas pressure vs. the flow rate. A dc motor connected to the pressure regulator (ZX 40XT, ITT, The Netherlands) adjusts the pressure as determined by the flow function and the calibration curve. The second method uses a pressure transducer (LX 1710G, National Semiconductor). The pressure is measured continuously. The signal from the transducer is converted from analog to digital form and is then compared by the computer with the value set by the flow function. When a difference is detected between the actual and the set pressure, a signal is generated. After amplification the signal activates two relays regulating the dc motor connected to the pressure regulator. When the pressure has reached the desired value, the motor is turned off by short circuiting, which ensures an instantaneous stopping of the motor. The computer program is structured in blocks. The flow function parameters and the final retention time, i.e., the time alloted to the separation, can be entered in one block. The computer then calculates the programmed final retention flow rate and the final retention volume and shows the graph of the flow function. In another block of the program, retention times are calculated from the known retention volumes of the analytes using eq 2. The results are presented on the screen as a simplified chromatogram. During the run the flow rate is calculated from eq 1 and displayed continuously vs. time. The actual values of time, pressure, retention volume, and flow rate are also shown on the CRT as well as the graph of the flow rate vs. time calculated from eq 1. RESULTS AND DISCUSSION The gas flow through the column is regulated by the inlet pressure. When the flow rate is expressed as an average flow 0 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO, 13, NOVEMBER 1985
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rate, the relationship between the inlet pressure and the flow rate (2) is approximately linear. By multiplying with a calibration factor, the flow function can he converted into a pressure function. Previous flow programmers (3)have been time-consuming to program. However, with a microcomputer control unit changes can be made quickly. The present flow programmer was designed mainly for studying the influence of various parameters in the flow program on the cluomatograms. It is easily adapted to different types of chromatographic equipment. The procedures included for calculating retention times, etc. greatly add to the usefulness of the programmer. From these calculations the approximate shape of the chromatogram can be predicted beforehand and the preliminary set of flow function parameters can be changed if necessary. Another improvement is the use of a pressure transducer in a feedback system since it provides better control over the change of flow rate. In the following examples the separations are obtained with flow programmed elution. Various injection and detection systems have been used, illustrating the versatility of the controlling unit. Three separations of standard mixture of organophosphorus pesticides are shown in Figure 2. In the first chromatogram, Figure 2A, the flow and the temperature were constant except for the steep initial temperature increase which occurred in connection with the splitless injection. The same mixture was then flow programmed, Figure 2B, selecting a relatively large range in the flow rate. The flame photometric detector worked very well in spite of a relatively great change in the flow rate during the run. The gain in signal to noise ratio was about 8-fold and the separation time was halved when compared with the nonprogrammed run. Moreover the peaks have almost constant widths and are more evenly distributed. The flow-programmed separation, Figure 2B, was run a t a temperature 50 aClower than the temperature-programmed run, Figure 2C. As illustrated with the separation of the pesticides, Figure 2B, the parameters of the exponential flow function can be chosen to give a steep increase in the flow rate at the end of the program. By so doing, only the very late components will he affected by the decrease in efficiency caused by the high flow rates. The effects of temperature and flow programming when applied in succession are shown in Figure 3. This method can be very useful when the maximum temperature for the stationary phase or the components is reached quickly. Figure 3A shows a temperature programmed separation of polyaromatic hydrocarbons (PAH) which lasted almost 1'/% h. In Figure 3B the temperature program is followed by a flow-
t,/min
50
0
10
'0
20
t,/min Flgure 2. A standard mixture of organophosphorus pesticides: 1, tiazinon; 2. m-parathion; 3,fenchlorphos; 4,fenitrothion;5, parathion; 6 . trichioronate; 7, tetrachlorvinphos; 8,ethion; 9. carbophenothion. During the spliiless injection the temperature has been increased suddenly from 120 to 200 "'2. Conditions were as follows: injector. splitless; column. AmAc, 25 m. 3.1 mL; carrier gas, nitrogen; detector, FPD. (A) Separated under isothermal and isorheic conditions. The curves show the flow rate (-1 and the temperature (---). (6)Flow programmed separation. The final flow rate was 25 mllmin. (C) Temperature programmed and isorheic separation.
programmed separation of the high-boiling components. Coronene, peak no. 17, was not eluted in the first run, Figure 3A, but use of flow programming makes it possible to elute it in about 1h, Figure 3B. Since a 50-m column is used for this separation, the flow rate can he increased only 3-fold before excessive pressure drop occurs. The falling needle injection system was used. Because of the low volatility of some of the components, the initial temperature is 200 "C whereas the starting temperature of the program is 150 "C. As a consequence, the temperature drops during the first few minutes. With this somewhat unusual temperature program all components leave the needle tip at the same time. Figure 3C shows a chromatogram of a sample of a spice mixture containing PAH,separated with the same program as in Figure 3B. The sample of Baltic herring was chosen because of its complexity. In Figure 4A the separation is isorheic and isothermal except for the fast temperature increase which oc-
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