CA1340390C - Capillary electrophoresis - Google Patents

Abstract

an apparatus is disclosed for providing capillary electrophoresis, which includes an electronically controlled valve system for automatically introducing a sample into the capillary by means of a vacuum at the end of the capillary tube. This approach of sucking in the sample is extremely accurate and reproducible, and results in a minimum of band broadening. Furthermore, it enables the entire capillary electrophoresis system to be easily automated. An automated temperature control system is provided which enables the temperature of the capillary tube (and hence the solvent/solute system) to be controlled during electrophoresis, thereby very directly controlling pH and electrophoretic mobility. In another embodiment, the capillary is prewashed and equilibrated to achieve substantially zero charge on the capillary wall, thereby essentially eliminating electroosmotic flow and substantially improving resolution.

Description

13~0390 METHOD OF PERFORMING CAPILLARY ELECTROPHORESIS

Backqround of the Invention This invention relates to capillary electrophoresis, or as it is more conventionally called "capillary zone electrophoresis" (CZE), and more particularly to automated methods and apparatus for introducing samples into capillary columns and for improving separations by using temperature control of said colu.~ns.
In recent years significant advances have been ~ade in micro-column separation techniques. A
principal advanta~e of such techniques is their sui~ability for analysis of extremely small sample volumes, eg. in the microliter or submicroliter amounts of sample. Bein~ able to analyze such small volumes has become exceedin~ly important with the explosion of research in the bioloaical field, because often-times biological samples are quite small.
One of the si~nificant problems with capillary techniques is in introducin~ sample into the capillary. One technique used in capillary electrophoresis, called sample injection, is electromigration, -a term collectively includin~ the effects of eletrophoresis and electro-osmosis ( See Jorgenson, J.W, and Lukacs, ~.D., J. Chromatogr~aphy, 1981, Vol. 218, pp. 209-216 Jorgenson, J.W., and Luk~cs, K.D., Science, 1983, Vol. 222, pp. 266-272:
and Wallin~ford, R.A. and Ewing, A.G., Anal.
Chem.,1987, Vol. 59, pp .68l-68q). In this technique, 13~0390 one end of the capillary and the electrophoresis anode are placed into the sample and a voltage is briefly applied, causing a small band of sample to electromigrate into the capillary. This method of sample injection suffers from discrimination within the sample because solutes with higher mobilities will preferentially migrate into the electrophoresis column , and therefore chanqe the relative composition of the sample. To avoid this problem, attempts to physically inject sample have also been reported (Jorgenson and Lukacs, Science, ibid). However these direct injection techniques cause band broadening, apparently due to the laminar flow profile introduced during the injection.
Other less common injection methods include qravity flow (See Tsuda, A., et al, J. Chromato~raphy, 1983, Vol. 264, pp. 385-392.), siphoning (See Honda, S. et al, J. ChromatoqraPhy~ 1987, Vol. 404, pp.
313-320.), and the usa of an electonic sample splitter (See Deml, M. et al, J. Chromatoqraphy, 1985, Vol.
320, pp. 159-165.). Each of these injection techniques are capable of placing subnanoliter volumes of sample into the electrophoresis column with a minimum of band broadening. However, the gravity flow or siphoning injection method is inaccurate and lacks precision in providing absolute volume amounts due to the unreliable position of the sample level which will change due to the sample withdrawal. The latter can only be neglected if the original sample volume is large compared to the volume injected. With the electronic splitter, a larger initial sample volume is required in order be able to split it down to the smaller size required for the column. Thus, some - 13403~0 sample may be wasted, or there may not be sufficient sample to perform a separation. Also, this latter technique is further complicated by requiring an additional controlled power supply or very careful control of the electric resistances in the different legs of the splitter. Furthermore, the need to use an initial larger sample size si~nificantly decreases the number of applications for which it can be used.
What is needed is a simple, automatable, sample injection technique that is suitable for microvolumes, is capable of providing accurate sample volumes, and which produces a minimum of band broadening.

Summary of the Invention In accordance with preferred embodiments of the invention, an apparatus is disclosed for providing capillary electrophoresis, which includes an electronically controlled valve system for automatically introducing a sample into the capillary by means of a vacuum at the end of the capillary tube. This approach of sucking in the sample is extremely accurate and reproducible, and results in a minimum of band broadening. Furthermore, it enables the entire capillary electrophoresis sytem to be easily automated.
The apparatus includes first and second reservoirs that are electrically isolated from each other for holding electrophoretic media, a sample reservoir located in proximity of the first reservoir for holding a sample to be electrophoresed, and a high voltaae power supply connected between the first reservoir and the second reservoir. A first pressure l3~03sa source of a first sas having a first known pressure, typically the ambient air pressure, is used for providing an environment for the first reservoir and the sample reservoir, so that electrophoretic media in the first reservoir and sample in the sample reservoir are under the first pressure. The apparatus includes a pressure reservoir for holding a second gas talso typically air) having a second pressure that is lower than the first pressure. A capillary tube is also included in which to electrophorese the sample. A rack system is provided for holding the first and second reservoirs, the pressure reservoir, the high voltage power supply, the sample reservoir, and for holding one end of the capillary tube in the second reservoir. A gas connecting system connects the second reservoir to the pressure reservoir, the connecting system having a valve for venting the connecting system to the first pressure source and for blocking communication of the second reservoir with the pressure reservoir while venting to the first pressure source. The apparatus also has an insertion element for inserting the other end of the capillary tube into the sample reservoir and into the first reservoir. In the preferred mode, the apparatus includes a computer system for controlling the insertion element and the valve, so that when the other end of the capillary tube is in the sample reservoir, the valve permits communication of the second reservoir with the pressure reservoir for a controlled period of time for sucking the sample into the capillary tube. Additionally, in the preferred mode, the computer system causes the other end of the capillary tube to be transferred to the first ~ 1~4~390 reservoir after the sucking of the sample into the capillary tube. After the sample has been introduced into the capillary tube and the tube has been transferred to the first reservoir, the electrophoresis is begun.
An additional important feature of the preferred embodiment is that an automated temperature control system is provided which enables the temperature of the capillary tube (and hence the solvent/solute system) to be controlled during electrophoresis. This is particularly advantageous in that for many buffers, the pH is a strong function of temperature; hence temperature control is very directly pH
control. Additionally the pH can have a direct effect on the electrophoretic mobility and hence the separation efficiency.
In another embodiment of the invention, the capillary is prewashed and equilibrated to achieve substantially zero charge on the capillary wall, thereby essentially eliminating electroosmotic flow and substantially improving resolution.
An aspect of this invention is as follows:
A method of performing capillary electrophoresis comprising:
introducing an electrophoretic medium into a capillary;
introducing sample into the capillary;
applying an electric field to the capillary to cause electrophoresis;
adjusting the temperature in the capillary to vary the pH to cause differential separations in the sample during electrophoresis;
detecting the sample introduced into the capillary.
Brief Description of the Drawings Fig. l show6 an apparatus according to the inventlon .
Fig. 2 i8 a table that illustrates the effects of capillary prewash on electroosmotic mobility.
Fig. 3 is a graph showing the results of Fig. 2.

-- 1 ~ 4 ~ i3 5a Fig. 4 is a schematic representation of a Functional Layout.

Descri~tion of the Preferred Embodiments Fig. 1 is a partially sectioned illustration of a 134034~

preferred embodiments of an automated capillary electrophoresis, henceforth CZE, apparatus according to the invention. In this preferred mode, the apparatus includes an environmental enclosure 11, which has access openings (not shown), and feedthrough of various kinds through the walls of the enclosure for elements that must be connected to elements outside the enclosure.
Electrophoresis is accomplished within the enclosure in a capillary tube 13, preferably constructed of fused silica, such as is typically used for high sensitivity liquid or gas chromatography. One end of capillary 13 is immersed for the process in a buffer solution 19 held in a first container 21, and the other end is immersed in a buffer solution 15 and in a second container 17. Buffer solutions 15 and 19 are typically the same solution, and many are well known in the art. The position of capillary 13 may be moved e.g. to replace buffer solution 19, using motor 65. Motor 65 may be used to rotate worm gear 71, which causes movement of threaded bo~s 73. Threaded boss 73 is connected to clamp 77 by rod 75 which may move between stops 79 and 81 on shaft 69. The presence of liquid in capillary 13 is detected by sensor 61.
Although in the prior art many different pH's have been used for the various buffers, depen~ing on the particular experiment being performed, in this preferred embodiment, it ha~ been found that for capillary electrophoresis that a relatively low pH is best. In the preferred mode, to achieve the best separations, the pH i5 adjusted to the point of zero electric charge of the buffer-capillary combination, ie. the point at which there is no charge on the capillary wall. As will be discussed subsequently in more detail, the point of zero charge will vary depending on the buffer used and the pretreatment of the column.
However, as a practical matter, typically a pH below about 2.5 will suffice. Also, as will be discussed subsequently, sometimes particular buffers are used which are temperature dependent, ie. their pH
A

.
~7~ 1340~0 varies strongly with temperature, or stated another way dp~/dT is relatively large.
~ iew 23 is an enlargement of capillary tube 13 in cross section. The internal diameter of the capillary, D1, varies for different kinds of samples and for other reasons. A typical value for D1 is 0.05 mm, and aenerally varies between zero and 200 microns. The wall thickness of tube 13 is small enough that the tube is flexible and and may generally be manipulated without breaking. Also, the small diameter allows for effecient heat transfer.
Second container 17 has an airtight top 25.
Capillary tube 13 enters the second container through a stopper 27 maintaining an airtight seal. There are two additional penetrations through top 25. A hollow tubing 29 enters through stopper 31 and and an electrode 33 enters throu~h another stopper 35.
Stopper 35 is typically of an electrically non-conducting material. From electrode 33, an electrical lead 37 goes to an electrical feedthrough 39 which allows an electrical signal or power to cross the wall of the enclosure without shorting to the enclosure. On the outside, electrical lead 41 goes to a terminal of a hi~h voltage power supply 43.
From the opposite terminal of power supply 43 another electrical lead 45 goes to another feedthrough 47. Inside the enclosure lead 49 goes to an electrode 51 immersed in buffer solution 19 in first container 21. With buffer solution and sample material in the capillary tube and the tube ends immersed in buffer solution in the two containers, power supply 43, through the electrical leads, feedthroughs and electrodes, may be used to maintain an electrical I D
-8 1 3 4 0 3.9 0 potential across the ma~eria~ sn t~e capillary tube~
The se~nd con~ainer rests on ~ ~upport 53 wi~h anelectr~ ~al insul~tor 55 ~4t~e~n the con~ainer and the s~pport. ~h~ ins~lator is ne~de~ if the ~ontainer and support are electrically conducti~e. Pirst container ~1.
rests on a movea~le, sliding support 57 ~nd ~n insu~ator Sg .
A dct~ctor ~1 is positioned relative to the capillary tu~e to measure the results of ele~trophoresis in tne capil~ary. such detectors are ~el~ ~nown in thc art, ~nd include for exa~ple an AppliQd R- osyste~s ~odel 7g3 W ~Visi~le D~tect~r, which is a variable wavelength ~rogrammable detec~or ~hat is specifically adapted for on-column detec~ion. Ele~trical leads throug~ ~edthroughs ~
carry power and signa~s ~r the in~ru~Qnt. There may be more than the two leAAs shown.
When the elec~rophoresis process is complete on one sample, and another sa~ple is wanted in the capillary for analysis, a new sa~ple may bo loadQd without manual in~erventivn or dis~ur~ing the environmental enclosure.
motor ~5 po~ered by leads through feedthro~gh 67 and su~or~ed by bracket member fi~ may be a~ti~at~ to ~urn lead screw 71. Nut 73 is a~ta~hed to ~emher 75 with a clamp -/7 se~urely hold~ng tube 13, so that tu~ning lead 25 ~crew 71 will raise and lower the tu~e by the distance be~ween stops 7~ and 81~. This distance is se~ ~ b~
s~f~ic~ent for the lower end of capill~ry tu~e 1~ to be raised abo~e ~ne ri~ of con~ainer 21, and lowered again.
With tub~ 13 raised above the rim of container 21, 30 mo~or 83 may be acti~ated by leads thro~gh feedthr~ughs 85 ~o turn lead screw 87 mo~ing sli~e 57 13403~0 along support 89. A sample container 91 with multiple microvolumes such as 93, arranged in a row in the container, is prepared in advance and placed adjacent to container 2~ on slide 57. Each microvolume may contain a sample to be analyzed. Typical injection volumes range from 1 nl to 10 nl in this preferred mode, although other size samples could of course be chosen depending on the size of the reservoir used to hold the sample and the size of the column. By controlling motor 83 moving slide 57, using worm gear 87, microvolumes 93 of container 91 may be moved to be directly below the end of capillary tube 13, which may then be lowered into the microvolume by control of motor 65, as discussed above. Once a new sample is drawn into the capillary, the capillary may again be raised, container 21 returned to position, and the end of the capillary re-immersed in the buffer by lowering the tube.
To inject a new sample, while one end of the capillary is in one of the microvolumes of sample material, a relative vacuum is drawn in second container 17 by means of tubing 29 which exits the environmental enclosure. Motor 95 is controlled to rotate a three-way rotary valve 97 opening tubing 29 to vacuum reservoir 99.
The reservoir is maintained at desired vacuum level by vacuum pump 101 through isolation valve 103. A vacuum sensing gauge 115 with programmable signal points monitors the vacuum level in reservoir 99. The pump is powered by motor 105. Careful control of timing and vacuum level provides a very accurate method for drawing a predetermined amount of sample material into the capillary, as well as other benefits. As an example, using a pressure 13403.g3 differential of 5.0 in. of Hg between the vacuum reservoir and the enclosure 11, with a 65 cm long fused silica capillary having a 50 micron inside diameter, a 2 second open time for valve 97 results in an injection quantity of 5 nanoliters of an aqueous solution.
Another important f eature of the apparatus according to the invention is that the temperature inside the environmental enclosure 11 can be controlled. A heatin~ element 107 is powered through feedthroughs 109 to provide heat, and a heat sensing element 111 monitors temperature through leads 113.
As will be discussed subsequently, such provision for temperature control is very useful, since some buffers have a temperature dependent pH, and for those buffers pH can be controlled automatically by controlling temperature. Temperature control is also useful in the more general case since other kinds of variations are avoided if a uniform temperature is used throughout a separation. For example, viscosity and therefore mobility are most often strong functions of temperature, so that for reproducability, temperature control is requiret.
Power and control leads for all the electrical equipment associated with the apparatus of the preferred embodiment are carried by electrical conduit 121 to a control interface 119 which provides power terminations and switching of signals for control purposes. The control interface is connected to and manipulated by a computer 117 which can be pre-programmed so that critical parameters may be maintained and sequences of analyses may be performed automatically by the apparatus. For example, the 13~03~90 vacuum level desired can be entered as control data, and the computer, through the control interface, monitors the signal from vacuum gauge 115 and opens and closes vacuum isolation valve 103 so that the desired vacuum level is closely maintained. As another example, the computer can be used to control the temperature inside the environmental enclosure by monitoring temperature sensor 111 and controlling power to heating element 107 as needed to maintain the programmed temperature. Also, the computer can be programmmed to allow a se~uence of analyses to be made, using the several samples preloaded into microvolumes in container 91, controlling the electrical devices in the required sequence. The program may be set to run analyses on all of the microvolume samples, one-after-the other, or to allow for manual intervention and initiation between each analysis. Another important feature of computer control is that the computer makes it possible to reverse polarity of the capillary electronically.
Hence, for solutes that are of opposite charge, one can reverse the direction of migration of solute particles and thereby use the UV detector at its fixed location. Details of the program structure for the computer are provided in Appendix A.

Control of pH
In capillary electrophoresis in free solution and in some gels, solutes with different charges (absolute) have different electrophoretic mobilities and can therefore be separated. Separation efficiency can be improved if the selectivity (ie. the relative difference in electrophoretic mobility) between two or ~ 13 10390 more solutes can be changed during the eletrophoretic run. One way to achieve this is to change the relative difference in the effective charges on the species to separated. In many cases the pH (or better, the pK) of the solution in the capillary, which is mostly buffer, determines the charge on solutes that obey the rules of acid/base equilibria.
For e~ample, for CHES (Cyclohexyl amino ethane sulfonic acid) in water, a chemical equilibrium is established which is dependent on the particular temperature, as indicated by the following formula:
O -N CH~-CH~-SOO- +OH- = O -N-CH.-CH -SOO- +H~O
ZWITTERION ANION
PX(20 C)-9 55 at pH=9.55, 50% of the CHES is in the zwitterion fcrm and 50~ is in the anion form. By increasina the pH to 10.55, the anion (RSO3-) concentration will be ten times that of the zwitterion and by increasing it to 11.55, the equilibrium will be pushed almost completely to the anion side. At that point only 1%
of the CHES will exist as a zwitterion. The anion will posess a certain electrophoretic mobility while the zwitterion being electrically neutral will not have an electrophoretic mobility. What this means is that at a pH of about 7.55, the CHES solute will have practically no electrophoretic mobility and at a pH of about 11.55, it will have nearly the mobility of the anion. Hence, by changing the pH of the solution, the mobility of a solute can be changed.
As indicated earlier, in many cases, the pH ~or pK) of buffer solutions are a function of their temperature, and different buffer solutions have different temperature characteristics. By changing the temperature of the buffer in time or in space, different pH's in time_and in space can be generated and thus the mobility of a species can be manipulated. In general, the pK of a solution is given by:
pK = pH - log[RS03-]/[R'SO3-], where [RS03-] corresponds to the concentration of the anion RS03-, etc., and in many cases the pK has a strong temperature dependence. As an example of how to use this temperature dependence during the performance of a separation, suppose that a separation is to be performed on a solution containing three solutes, A, B, and C, and that the A and B separation is best performed at a first temperature Tl with A coming through first, and that the B and C separation is best performed at a temperature T2.
The separation is run for a first time at temperature Tl, until A is separated from B and C, then the temperature is changed to T2 until B and C are separated. Similarly, more complicated temperature profiles can be used depending on the particular solutes being separated, for example continuous programming can be used if desired.
It should be appreciated by those skilled in the art that solutes that are to be separated may also obey the acid/base equilibria rules, and as a result also can change their degree of ionization with temperature. This solute effect will be superimposed on the pH change of the solvent (buffer) and hence, depending on the choice of buffer and solute 134039~

combination, can provide an enhanced mobility difference, decreased mobility difference, or no change in mobility difference at all. Hence, various combinations of buffer and solute should be chosen to achieve the desired effect.
As a specific example of the effects of varying temperature, and hence pH, an experiment was conducted to investigate the relative electrophoretic mobilities of two proteins, Myoglobin ~wsm) and Myoglobin (hh).
A fused silica capillary was used having a 55 cm length to the detector, a total length of 70 cm, and an inside diameter of 0.050 mm. Using a 10 mM
Tris-HC1 buffer, and a 20 kV electric potential, the change in relative difference in electrophoretic mobilities (ie. selectivity) of the two proteins was measured between the temperatures of 26.9 C (pH=8.90) and 62.4 C (pH=7.90), and was found to be minus 45%.
As described earlier, another important aspect of the invention in achieving a high selectivity, particularly in protein separations, is to eliminate charge on the capillary wall. The purpose is to eliminate electroosmotic flow, so that the column is not being swept during the run, thereby providing a longer separation time in the column (lower average velocity of the species to be separated), and hence better resolution. Also, by eliminating charge on the wall, positive ions (eg. proteins) do not stick to the wall, unlike the typical case when the wall is negatively charged. One way to achieve zero charge on the wall is through control of pH. Generally, the electroosmotic velocity is proportional to the zeta potential times the applied electric field divided by the viscosity. The zeta potential describes ~- 13~039d electrostatic forces in the interfacial double layer between two phases and is, among others, a function of the differential adsorption of ions. When there is no electroosmotic flow, the zeta potential is zero, and there is no charge on the capillary wall. Hence, by measuring the electroosmotic mobility, ie. the electroosmotic velocity divided by the applied field, as the pH is changed to achieve zero mobility, the point of zero charge on the wall can be determined.
An example of the effects on electroosmotic mobility resulting from pH changes and some surprising results from differences in capillary preparation are illustrated in the Table of Fig. 2, and in Fig. 3. In these experiments, a number of buffers were used in order to cover a wide range of pH levels, since as a general rule any one buffer has a relatively limited range of pH values over which it is useful. The capillary used was a fused silica capillary supplied by Polymicro Technologies, and had a length to the detector of 30 cm, a total length of 50 cm, and an inside diameter of 0.050 mm. The applied electric field used was 360 V/cm. Except for run 4, where the order of preparation was reversed to check for reversibility (ie. steps 4, 5, and 6, preceeded steps 1, 2, and 3 below), the protocol for capillary preparation was as follows:
1. wash capillary with 1 M NaOH for 3 minutes;

2. equilibrate with buffer for 5 minutes;

3. run with mesityl oxide as a neutral marker and measure elution time for the marker;

4. wash capillary with 1 M HCl for 3 minutes;

5. equilibrate with buffer for 5 minutes;

6. run with mesityl oxide and measure elution 1~ 103~d time.
The buffers used were CAPS (ie. 3-(cyclohexylamino) propane sulfonic acid), BICIN~ (ie.
N,.~-bis (2-hydroxyethyl) glycine), MES (ie. a sodium salt of 2- (N-morpholino) ethane sulfonic acid), and citric acid.
A graph of the electroosmotic mobility, shown in Fig. 3, illustrates dramatically the results of lowering the pH. Clearly, as the pH is lowered there is a sharp decrease in mobility indicating that the charge on the wall is quickly approaching zero. For NaOH treated tubes, a pH of below about 2.5 corresponds essentially to zero electric charge on the wall (the baseline noise is proportional to the current, so as a practical matter it is important to use a low current, and low pH buffer). Even more striking, however, is the effect of the prewash.
Although NaOH is the quintessential strong base that is typically used for washing glass, it is clear that the US2 of a strong acid such a HCl is a much better prewash if the purpose is to eliminate charge on the capillary wall. For example, if the capillary prewash is performed with HCl, a pH of about 4.0 eliminates about 97% of the charge on the capillary wall, and produces an electroosmotic mobility that is even lower than that achieved using a pH of 2.5 if the prewash is with NaOH. Furthermore, it appears that the effects of the different prewashes are substantially independent of each other, since in run 4 where the order of the two prewashes was reversed, the results are substantially the same. As a practical matter, it appears that removing 95% or more of the charge on the capillary wall is important in performing high 4039d resolution capillary electrophoresis, regardless of the prewash that is used. However, it appears that eliminating that charge is much easier to accomplish, and allows use of a much higher and more easily attained pH level, if the capillary is prewashed with an acid instead of a base before the run.
It will also be appreciated by those skilled in the art that there are several ways to control the temperature of the solvent/solute system. For example, one way has already been described which uses a heater system for environmental chamber 11. Another approach would be to use one or more electrical heaters wrapped around the capillary tube, and another would be to use one or more pieces of insulating wrap on the capillary tube. Those skilled in the art will undoubtedly be able to think of other equivalent methods for controlling the temperature to effect electrophoretic mobility. Those skilled in the art will also understand that in some instances it may be preferred to not have all components inside the enclosure 11. For example, the detector sometimes may be located outside the enclosure along with the corresponding portion of the capillary where the UV
detection is to take place. Such an approach would facilitate service of th UV detector system. Also, instead of raising and lowering the capillary, one could raise and lower the sliding support to insert and remove the capillary from the sample and buffer reservoir. It should also be apparent that one could use electrophoretic media other than aqueous solutions, for example organic fluids could also be used, a specific example being acetonitrile.

,, . 134~3~0 A software specification for a preferred embodiment of the invention is described below. The specification includes an operational description, methods of operation of aspects o~ the software and examples of the hardware/software interface.

13403.9o ' L~ Operational DescriDtion:
The software will have three primary operating mode~. These ~re I~ un, and ~ ~ual. Each of these modes relates to a basic st~te o~ operation for the instru~ent. Each of the primary modes have a series of associated sub-modes.
Functional T~ out: ' . The functional layout is shown in Fig. 4.

B. ~ode Descr~pt~ons:
Each Mode i6 selected by using the special function keys to the right of the di~play. Choices are presented to the cbemist on the d$splay and by using the NEXT and PREVIOUS keys changes the appropriate values in the appropriate units for the particular choice being edited or selected. To change from choice to choice the Left and ~ight Arrow keys are used.
The following are brief descriptions of the instru~ent's modes.
1. Tdle ~ode:
This is the power on and default operating mode of the instrument. Any of the other ~odes may be startcd from idle, and return to idle when they are exited. Idle allows the user to execute the functions listed below.
a) Cycle Edit b) Method Run c) Manual Cpntrol d) Configuration Doors may be opened in Idle mode at anytime.
a. ~it ~ethod:
Allows the chemist to display, and ~odi~y ~aved ~ethods or to copy a ~ethod into a new method na~e for editing. The system will maintain in ~ battery bac~ed up ~ RAM between 1 and 20 methods. The sVstem w$11 contain in ~OM exa~ple executable methods [ 1-20 wh$ch can be copied into RAM ~nd be edited.
This function w_ll allow the chemist to access ~ method, modify it and save it back. All method para~eters will be validated at input, and prior to sav$ng. The list of method parameters, and their meaning will be presented later.
Edit$ng of any RAM based method can occur during a method run but will not change the ~ethod run in process. Changes made to the ~ethod in process will be seen the next time that method is run.
A

Hethn~
Allows the chemist to execute one of the saved methods automatically. Execution perfor~s the following set of cycles, in the order shown. Doors can be opened only when a pause state is ~n process. Wi~h the combination of RAM and ROM methods there will be a total of 40 methods available to the chemist.
i) Interlocks All interlocks are verified.
ii) Wash Capillary washed w/ reagent.
iii) Equil Buffer Bu~fer is brought into the capillary and equilibrated.
iv) Marker In~. Marker solution in~ected.
v) Sample In~. Sample solution in~ected.
vi) Detector Initialize Detector vii) tO Initial Time Step.
viii) tl First Change option.
ix) t2 Second Change Option.
x) t3 Third Change Option.
xi) tEnd Run Run continues until this time expires, or stop/abort pressed.
ç~ Manual Control:
In this mode the chemist can set instrument parameters from the key board with realtime results. Introduction of solutions from vials is allowed, but the sequence of solution introduction is carefully controlled when ~IGH VOTT~ S are involved. High voltage is allowed only when capillary is in a sample or buffer position, and all safety ~nterlocks are verified. Doors may be opened in the manual mode when no voltage is applied to the capillary and the vial holder is not ~oving.
d~. Conf~gurat~on:
~ his allows the chemist to set default values for the system parameters. This includes all detector parameters.
Self test is e~ec~ted at power up doing only those tests that do not require user intervention, and whenever diagnostics are required all tests are available.
Adjustment of capillary is accomplished at this state allowing the vial holder to be in the up position with the doors open.

21 134g3.90 .

ethods: -DescriDt~on of Para~eters:
The ACE will be capable of running up to four (4) samples unattended by the-c-hemist. To do this t~e system must kno~ in advance the values for a set of control parameters. Shis set of parameters will be called an A OE ~ethod.
The following is a list of the parameters in an ACE method.
1' Wash Time - 2 Buff Equil Time 3 Marker In~ Time 4 Sample In~ Time Polarity 6 Wavelength 7 Range 8 Risetime 9, Autozero 10' Time 11 Buffer Number 12 Voltage 13~ Temperature Each of these parameters is used durin~ an automated run to control the instrumental conditions. The last four parameters are optionally changeable three times ~uring an AC~ ~ethod run.
Some of the parameters are not currently set by the chemist, but use instead preset values determined by ABI/SC chemists.
1' Temperature Eguil Time 2 In~ect Vacuum Level 3~ In~ect Voltage 1~ Wash Ti~e:
Range: 0 - 60 min Default: 5 min Unit: l min The wash time is how long a strong buffer is applied to the capillary as the flrst step in an automated run. If this time is set to zero, this step in the run sequence will not occur.

A

2. ~ffer ~qu~l~brat~on Time:
Range: 0 - 20 min Default: 1 min Unit: 1 min The third step of the run procedure is to introduce the buffer into the capillary. This is performed by a vacuum sucking on one end of the capillary. $his time is variable, and can be set by the chemist. Temperature equilibration could take place at the same time possibly. If this time is ~et to zero, this step in the run sequence will not occur.
3. ~arker T~ect~on T~e:
Range: 0 - 30 sec Default: 1 sec Unit: 25ms accuracy .5 second increments The marker solution is introduced into the capillary at the end of-the equilibration state. A very small amount (a few ~L) of this solution is introduced as a plug into the end of the capillary, by using a lower vacuum for a controlled period of time.
4. SamDle Tniection T~me:
Range: 0 - 30 sec Default: 3 sec Unit: 2Sms accuracy .5 second increments The sample solution is introduced next into the capillary as a second small plug. Again the volume injected is a function of ~acuum level, time, viscosity and capillary dimensions.
5. Polar~t~:
Range: A ¦ C
Default: Anode 1 at sampling side Unit: N/A
The polarity of the sample side buffer vial can be either positive or negative. This value is set in the method and is maintained for the length of the run.

6. ~A~el ength Range: 190 - 700 nm Default: 215 n~
Unit: 1 nm The absorbance wavelength of the detector for the run. This value is user set at the start of the run, and remain constant through out the run.
7. Range:
Range: 0.001 - 3.000 AUFS
Default: 0.01 AUFS
Unit: 0.001 - 0.1 is 0.001; 0.1 - 3.000 ls 0.01 AUFS
The absorbance units full scale o~ the detector is user set at the start of the run and remains constant for the duration of the run.
8. ~seti~e:
Range: 0.02 - 5.0 secs Default: .5 Unit: 8 steps The detector risetime (response time) is user set at the start of the run, and remains constant for the duration of the run. The value has 8 incremental values between 0.02 and 5.0 seconds.
9. Autozero:
Range: Yes/No Default: Yes Unit: N/A
For the present system the detector will be autozeroed at a set time after the voltage is turned on in the run. t See T Zero sequence ~
10. ~Ç:
Range: 0.0 - 60.0 mins Default: 25 m~ns Unit: 1 ~in The time along with the voltage, and temperature will be 134039~

used to create a 6eries of steps for the run to follow. This will ~e defined in the next section.
11. Buffer Number:
Range: 1 --2 Default:
~ here are two (2) buffer positions on the vial tray. Each method will need to know wbich buffer(s) is to be used with the run. ~ Both buffers can be used in the sa~e N n. 1 When buffer chanqe is selected during a run the software will select the opposite buffer automatically the user will not need to specify a buffer number. When ~ buffer cbange is selected the voltage will be turned off during the change and will be turned on.

12. Voltage:
Range: 0 - 30 XV
Default: 15 KV
Unit: 1 XV
The value of the voltage is the potential across the ends of the capillary at the time specified. This ~alue may range from 0 to 30 Xilovolts.
13. Tem~erature:
Range: 15 - 60-C
Default: 30-C
Vnit: l-C
~ he value of the temperature is the temperature of the thermostated oven in the instrument at the time specified. It may range from 15 to 60-C. I~ the value entered is below 25-C
than a caution will be displayed that coolant must be present to achieve desired temperature. In the future the software with a hardware ambient temperature detector will give this caution according to the temperature selected and the actual ~mbient temperature.

131039d xed Parameters:
Some parameters are not currently set by the che~ist, but use instead preset values determined by ABI/SC chemists.
Temperature Eauilibration T~me:
Future Range: O - 10 min Fixed: 6 min The primary step of an automated run is bringing t~e oven up to the runtime te~perature. This value is set in the software, and cannot be changed by the chemist. If temperature is achieved prior to expiration of this time the run procee~C to the next step.

2. Tnject Vacuum ~evel:
~ange: O - 20 inches Hg Default: 5 inches Hg Future Unit: 1 inch The vacuum level which $s used to introduce the sa~ple, and marker into the capillary is set in the software; and copied into the method when $t is f$rst created.

3. ~n~ect Volta~e:
Range: O - 30 XV
Default: 6 XV
Future Unit: lXV
The voltage level at wh$ch sample and marker $s electrophoresed $nto the capillary is set in the ~oftware A

26 13 40 3~0 ~e~h~ ion:
Section A above describes the instru~ental parameters involved in an automated run performed on the ACE. This section will briefly describe cycle particular information and operation.
When the chemist selects the RUN method function, the software asks which method to run. A number between 1 and 20 is entered for the desired method. That method is then copied from the method buffers into the run buffer. At this time the chemist is asked the number of Samples to analyze using this method. The instru~ent then begins execution of the run buffer.
Temperature ~nU~l~hration:
An optional Pause can be performed before this step continues. ~ See Pause Note ]
The second state in a run sequence is temperature equilibration. Oven is to start equilibration as soon as value is entered ~ Manual ~ode ~ or as Foon as a method number is selected ~ Auto Mode ~. The oven temperature is set from the value in the time zero (0.0) step of the method. A period of up to six minutes is allowed for the oven to reach this temperature.
The temperature is determined to ha~e been reached when:
10 t to be deter~ined in actual use most likely a value of 2 1 ceguentially acquired temperature ~alues have delta between t~e set ~alue, and the read ~alue of + .l-C precision; + .2-C accuracy; read + .25-C for 10 Secc!nAc When this condition is met, the run advances to the next state.
pash:
An optional Pause can be performed before this step continues. 1 See Pause Note ~
The first state in a run sequence is the WASH state. This state draws a strong acid or base solution throuqh the capillary.
This is done by positioning the capillary in the wash vial. The capillary is then exposed to a vacuum at the wash fixed le~el.
This exposure has a time duration as set in the wash time.

~ 27 13 40 390 1 ~uffer ~guilihration:
An opt~onal Pause can be performed before this step continues. ~ See Pause Note ~
The third state of a run is tbe equilibration of the buffer to the capillary. Shis is accomplished by moving the capillary to the buffer des$gnated ~n the method. The buffer is then drawn through the capillary at the designate wash vacuum level, for the designated buffer equilibration time.
4. Marker Tn~ection:
An optional Pause can be performed before this step continues. ~ See Pause Note ]
~ he ~ourth state of a run is the in~ection of the marker solution into the capillary. This is accomplished by positioning the capillary into the marker vial. ~he marker is then drawn into the capillary for the designated in~ection time. The method of injection is by exposing the capillary to a vacuum at the injection levei, or a voltage at the in~ection level.
5. Sa~ple Number:
Range: 1 - 4 Default:
There will be four (4) sample positions on the vial holder.
At the time a method run is selected the number of Samples to be analyzed will also be selected.

6. SamDle In~ection:
An optional Pause can be performed before this step continues. t See Pause Note ~
The fifth state of a run is the in~ection of the sample into the capillary. The capillary is positioned to the designated sample vial, and the processed the same as a marker, except the exposure time is that of the sample in;ect time.
7. Beq~n Final Run:
An optional Pause can be performed before this step continues. 1 See Pause Note ~
At this state actual electrophoresis begins. The polarity of the buffer is set per the method and the voltage is set to the level specified in the time zero (0.0) step of the method.
A specified number of ~econds after the voltage is turned on, the 1~ 103~0 detector ls ~ent the command to autozero itself, and the re~ote integrator event is executed.
Each method can have up to four (4) gradient steps associated with it~ These steps are contain the information shown below:
Time: Voltage: Temperature:
The method steps are executed in time sequential order. E~ery method has a time 0.0 step. This sets the initial voltage and temperature for the run. If no other steps are entered, these conditions are maintained for the length of the run. If other steps are present the voltage and/or temperature are stepped over the tlme period between sequent$al method steps. The time of the final step is the total runtime of the method. For instance the following method would have a runtime of 30 minutes, and would have two state changes ~ voltage & or temperature ].
Time: Voltage: Te~perature:
0.0 min 20 XV 30 ~C
10.0 30 30 1 empty 20.0 30 1 empty 40 30.0 30 40 7. ~nd of Run:
When the runtime has been reached, the ~oltage is turned off. At the same time the remote integrator event is executed again. The instrument then returns to the Idle mode. The temperature that was T zero temperature is maintained as the idle temperature before the next run.
8. Run Functions:
There are four functions available to the chemist during an automated run. The ~lrst of these is autozero. By executing this funct~on, the chemist sends the detector the instruction to autozero itself. The second function when executed stops the methodmed run early. The third function aborts the methoded run at anytime in the sequence. The fourth ~unction allows the chemist to go from automated control to manual control at the current conditions of the instrument.

- - 1340~3 Pause Note:
SemiAutomatic operation will follow the same sequence as the auto~ated operation with a pause at the beginning of the cycle.
After the operator has been pushed the proceed button the automatic sequence-~ill continue.

_ 30 13~0390 ~ ' C. CYcle Ed~tin~:
When a chemist executes the cycle edit function from the Id~e ~ode, he is first quiered for the method number to edit.
This method is t~en copied from the method buffers to the edit ~uffer. The editi~g screen/s are then presented to the chemist.
The chemist can move between the edit fields to change the various ~ethod parameters. The cycle parameters are presented to the chemist and by using the NEXT and PR~VTO~S keys changes the appropriate values in the appropriate units for the particular parameter being edited.
One function available during editing allows the chemist to save the changes back to the method bu~fer. When this function is executed, the entire method is validated, and then saved bac~
to its method buffer. The e~itor is then exited, and the instrument is returned to the Idle condition.
A second function allows the chemist to print a method out.
And a third function allows the chemist to modify the detector parameters.
1. Method Choices:
The following chart is a matrix showing the options and choices available when editing ~ run ~ethod.
Sa~ple Method Possibilites Cvcle Time Pause Volt Temp ~tem Other Wash X X N/A N/A N/A N/A
~uffer X X N/A N/A N/A N/A
Marker X X N/A ~/A X N/A
Sa~ple X X N/A ~/A X N/A
De.ector N/A N/A N/A N/A N/A Rise. ran~e. wave T ~ero O.O N/A X X BUF Polaritv T ~ X N/A X X BUF ODt ona_ T 2 X N/A X X BUF OPt ona_ T 3 X N/A X X BUF OPt ona T ~nd X N/A

~ 31 1340390 State Diagrau 8 9 lOll _ _ Get Method Read Hethod into tl _ t2 _ t3 -¦tEnd¦_ ~ Sa~ples Valid M~ S~ buffer/nterlocks Set Temp. to tO
~~~~ 'Begin ---~
v Interlocks on Pause Y/N
v v tO (Zero) Polarity v Turn on Voltages 7 AutoZero, Start Init. 2 ¦Wash twa~O V
v ' Pause Y/N
Setup RiseTime 3 ¦ Buffer Detector Range 6 Wave Len Pause Y/N
tbuf'~ V
~tsamp'~ v 5 Pause Y/N 4 c ~_____ Sample Marker tmark~~
Rey: t-time T-Temperature Notes:
Boxes 2-5 time decreases to zero for completion of task.
- Boxes 7-ll time increase to cho~n ~alue for completion of task.
Box 6 MnST occur w~thin 6 minutes with the temperature stable.

A

- 1340.~0 ~ethr~ ~e~Q~y:
The ACE instrument will have the ability to save four (4) methods in battery backed memory. This will allow the chemist to establish a set of methods and then ~aintain those methods even when the instr~ment is turned off. ~n addition to the four ~ethods, there wlll be two buffers to hold the method currently being edited, and the method currently executing. This means there will a total of six (6) method buffers in the battery backed ram. A method buffer should consume approximately 30 bytes of memory.

, -- - 33 13~039i~

E~ CYC1~ Senuences:
The following section contains the sequence of events that are to take place ~or each operat~on. This is the start of the State definitions that the software will implement to create the desired use for the-~CE unit.
erl oc~a:

The following hardware interlocks prevent voltage from being applied to the capillary, rotation of the vial holder or up ~ovement of the vial holder.
a. Buffer Door b. Detector Door c. Vial Holder Door d. O~en Access Door e. Vial Holder in Place f. Vial Holder in Rotate allowed position When the Doors can be opened or must be closed is indicated in the next two lists.
Doors May be Open Doors Must be Closed 1. Idle Mode 1. Voltage applied to capillary 2. Manual Mode 2. Vial ~older Moving 3. Pause nterlock Seauence:
a. ~ook ~t Hardware Bit b. ~ook at Software Mirrors of proc~sse~ allowed 1. The software Mirrors are flags indicating the processes occurring at that ti~e. They get changed at cycle transitions, cycle operation and exit from the cycle.

._ 34 ii. ~nu~l1hr~te ~- .er~ture:
a. Read O~en ~emperature from A/D and store value b. Read Ambient Semperature and store value c. Compare Oven ~emperature to setpoint 1. If Reading ~ setpoint a. Delta Temperature > +15~C and ambient ~ Oven then open flap ~ turn off heater. Otherwise ~ust turn heater off.
- d. If Reading ~ setpoint then close flap ~ turn on heater.
Note: Check~n1 ambient temperature and giving cautions for temperatures close to or lower than ambient vill be a future design choice. Also in the future a val~e to control coolant flow will be under software control.
iii. Vacuu~ Wash Sequence:
a. Move Vial Holder to Wash solution b. Make sure Sn and In~ect val~es are closed c. Open 20~ valve d. Open In~ect valve e. Wait X time t user selected time f. Close In~ect val~e g. Close 20~ valve au~l~hrateBuffe~:
a. mo~e vial holder to designated buffer number b. Vacuum buffer c. Wait X time t user selected time - 13403~d . ~arker Tn~ect Seguence:
a. ~ove vial holder to marker solution b. perform user choice of ~acuum or ~oltage c. Wait X time ~ user selected t~me .b.l Marker VACU11~ Tn~ect Sequence:
a. Make sure 20~ and In~ect valves are closed b. Check 5" ~acuum level c. Open 5" valve d. Check 5" vacuum level e. Open In~ect valve f. Wait X s?con~ t 0 - 30 seconds user selectable g. Close In~ect val~e i. Close 5" val~e ~. Ad~ust 5~ ~acuum level v.b.~ ~arker Voltaae Tn~ect Seouence:
a. Check Marker position b. Check Interlocks ~ in Up po~ition c. Wri~e to D/A voltage des~red 1 Fixed ~alue at this ti~e~
d. Read A/D for current to indicate ~oltage conducting vi. Sa~ple In~ect Sequence:
a. move vial holder to sa~ple nu~ber desired b. perform user choice of vacuum or voltage c. Wait X time ~ user selected tl~e ]
.b.l Vacu~ Sa~Dle ~n~ect Sequence:
a. Make sure 20" and In~ect valves are closed b. Check 5" vacuum level c. Open 5~ valve d. Check 5" vacuu~ level ' e. Open In~ect valve ~. Wait X seconds t O - 10 seconds user selectablQ 1 g. Close In~ect valve i. Close 5" valve ~. Ad~ust 5" vacuum level vi.b.~ Voltaae Sample ~n1ect Sequence:
a. Check Marker position b. Chec~ InterlocX~ L in Up position c. Write to D/A ~oltage desired t Fixed value at this time]
d. Read A/D for current to indicate voltage conducting vii. Detector Seauence:
a. Set Wave ~ength b. Set Range c. Set Rise Time 37 1 3~ 03.9d vii~. t Zero SeouenCe:
a. Interlock~ OK
b. At Temperature c. Turn on Integrator d. Turn on Yoltage ~ Stepped ONLY 1 and ~tart run tlme e. Send Autozero command to Detector tl Seouence:
a. Go to next cycle i~ time i~ zero b. change voltage if non zero c. change temperature if non zero d. change buffer if non zero ix. t2 Se~uence:
a. Go to next cycle ~f time ls zero b. cbange voltage i~ non zero c. change temperature if non zero d. change buffer i~ non zero t3 Seauence:
a. Go to next cycle ~ time is zero b. chAnge voltage if non zero c. ~h~nge temperature if non zero d. change buffer if non zero A

38 13~o390 ~i~ t ~n~ Seouence:
a. Turn o~f Voltage b. Set temperature to what it was at time Zero c. turn off integrator d. Decrement number of samples to run i. if number of ~ample - zero than go to Idle ~i. lf number of samples not ~ zero begin autorun on next cycle. The autorun restarts at the beginning of the method with temperature equalibration.

39 131~ .9i~

F. special~z~ S~uences:
The seguence~ $n th$s section are ones that are used by larger general ~equences. Specialized sequence~ that can not find a home elsewhere are included here.
.
1~ CaDillarY V~al holder Seouence:
The time from vial to vial i~ 1/4 of a second giving a worse case of 1 3/4 seconds to the next vial. The following chart shows the position~ and coding for each of the eight possible vials.
Direction of rotation is >
VIA~S Saml Sam2 Sam3 Sam4 Wa~h Buf2 Bufl Marker Rough LED.l x x x x Rough LED 2 x x x x Rough LED 3 x x x x Exact LED x x - x x x x x x .2 Rotate Seouence:
a. Sampler Down and at IN positlon b. Turn ~otor On c. Watch for Ro~l~h ~ED Pattern d. When Exact ~ED is ON turn Motor OFF
e. Mo~e Sampler Up . ~

. .

- 40 13~0390 1.3 Up v1al ~older Seouence:
a. check interlocks b. ma~e sure IN posit$on, not rotating ~ volts off c. mo~e up d. stop when up 1.4 n~wn Vial Holder Sequence:
a. Volts off, Vacuum off ~ interloc~s OK
b. Move Down c. Stop when indlcator shows it is down 2. Power On Seauence:
a.. Initialize peripheral components b. Init~alize ports c. Initialize ~acuum d. Idle position ~ial holder e. Set Semperature to 30- C
f. display Idle mode screen 3. Self Test Se~uence:
a. Choice run A test or Auto run tests b. Run test(s) c. ~eport result(s) d. Wait here for operator to go back to Idle ~enu 41 ~ 1~ ID390 4. Po~ a~l Seouence:
a. Save time occured b. Save system status if needed c. Turn off voltage, ~acuum, motor movement d. Wait for power to be restored e. query operator for choices of recovery S. Real T~e Clock Sequence:
a. get command to be passed to chip b. send command c. get response if needed 6. Pr~nter Seauence:
a. Get pointer of data to be printed b. Init$alize pr$nter c. Get format for th$s data to be printed d. Start print data in proper format e. Complete pr$nter operation 7. ~f~er Bottle status Sequence:
a. Increment Buffer i L 2 number of times used counters b. If near depletion than give gentle caut$on c. If at or past depletion give firm warning d. reinitialize values as appropriate.

42 ~ 1340390 8. Ad~Ust 5~ Vacu w Seouence:
a. Read Vacuu~ A/D
b. Compare to setpoint c. if reading > setpoint - XX then open bleed down valve.
d. if reading ~ setpoint ~ XX then open ~leed up valve.
Pover Down Seauence:
a. query operator for Choices b. save status according to operator choices c. put vial holder in store position.
d. Give operator OK to power down.
10. Tnterrupt Service Routine:
a. Check Hardware Interlock bit b. Increment Timer Counters c. Future read ports to see they are at state desired 11. Rol~n~ Rob~n T~s~:
a. Software Interlock M~rror Check ( Future wben can read ports) b. Voltage t~6k 1. Read A/D and convert to system value 2 Ad~ust Voltage as needed c. Vacuum task - d. Temperature task e. Absorhanço task f. Status d~splay update g. Chemistry h. Run Setup i. Edit ~. Print k. RS-232 1. Manual Operation A

44 1 3~ 0 390 III. Manual CGI~O1:
Manual control allows the chemist to perform any of the cycles of an automated run, but with complete manual control of the process. ~his will allow the chemist to test new ideas, and conditions in a manner where instant feed ~ack to the changes are desired.
Manual Control will also have access to sub-state operations that are normally buried within an automated cycle. An example of such sub-cycles are the vial holder movement operations.
Manual control can be entered ~rom two distinct places. ~he first place is the Idle mode. There will be a function ~ey defined as manual control. The second place is from the Run mode. Again there will be a function ~ey (preferable the same ~ey) designated as Manual control.
When manual control is entered from the Idle mode, the instrument sets itself to the wash state o~ an automated run, but does not begln execution of the cycle. It waits for the chemist to specify which cycle it should execute, and what the condit~ons are.
When manual control is entered from the Run mode, the instrument continues r~nn1ng the current cycl-. If the tEnd cycle has been entered, the voltage, and temperature are frozen, and the instrument continues to operate at those conditions, until either the conditions are changed or stopped by the chemist. The chemist may choose to abort or allow the current cycle to complete.
When in manual mode, the chemist may select a cycle to execute. The chemlst than enters values for that cycle's parameters. ~f all conditions for that cycle are ~et than it is executed. ~his lncludes, safety interlocks, positioning of capillary, and any other safety related items.

, ~ 45 13 10~390 IV. Con~our~t~on:
Configurat~on allows the chemist to do two things. T~ey can set the default values of instrument parameters and self test the instrument.
Default instrument parameters such as the detector parameters tbat do not change as often as other instrument values can be set here, and any time a default value for that parameter is reguired, the software gets from the system configuration.
The instrument self test allows the operator to force the instrument to perform lts power up diagnostics. These will be defined as they are deemed ne~ess~ry.

A

- 1~403.91~

V. Sy~te~ Screen~:

~dle Screen:
Line 1 Time TOD Abs Temp Edit Line 2 Volts Current Y Run Line 3 Line 4 Forty gage 1234S67890123456789012345678901234567890 t w~th Run ~ n v~ v~ess Screen:
~ine 1 Method "Na~e" Cycle "Name~ Main-Line 2 Temp XX Volts XX Run--Line 3- Time XX Buffer X Print Line 4 Temp: XX Volts XX End XX Time XX Status Forty gage 1234567890123456789012345678901234567890 C. Manual Screen:
Line 1 ~ine 2 Line 3 ~ine 4 Forty gage 1234567890123456789012345678901234567890 .

Vl. ~rror ~andli~q:
There will ~e visual and audible error response to all errors. Each cycle that has error checking will have a section for error processing which will include message display and correct~ve action~f possible and if not an appropriate shutdown procedure.
~ rror Screens:
Line 1 Interloc~s Faulty Check Doors ~ Sampler Line 2 Line 3 Line 4 Forty gage 1234567890123456789012345678901234567890 ~ 48 13 40 390 VII. ~ar~Yare/Soft~are Interface:
The information in t~is section will specify the relevant aspects of th- hardware that the software needs for interface to the hardware. This includes port addresses of de~ices, control values of devices,-and conversions for DACs, and ADCs.
AutosamDler ~ontroller:
Port: 73h Autosampler consists of a pair of DC ~otors that are used to position a device. These motors are turned on and off, and the position is noted by monitoring a set of sensors. The bit pattern of the sensors indicates the po-ition of the autosampler.

B. Detector Controller:
Port: 72h At this stage the Detector Controller consist~ of an ADC to convert the absorbance signal from the back of the 783 detector to a digital ~alue, and then display it. Other control of the detector will be via the Z80'~ RS-232C port.

49 1340~0 C ~emDerature Controller:
Port: 71h The temperature is controlled by reading the temperature value, comparing it to the setpoint value, ~nd then turning the heater on/off and the opening/closinq the flap to ad~ust the temperature up or down.
The following is the derivation of the temperature ADC
reading to a temperature $n ~C.
0 to S Vin - 0 to 60 ~C
255 bits - 60 ~C
1 bit - 0.253 ~C
(ADC value ~ 253) / 1000 - T ~C
0 to 5 Vin - 10- to 60 ~C
255 bit~ - 50 ~C
1 bit - 0.196 ~C
~ADC value * 196) / 1000 - T ~C
0 to 5 Vin - lS- to 60 -C per parameter temperature spec.
255 bits - 45 ~C
1 bit - 0.1765 ~C
(ADC value ~ 176.5) / 1000 - T ~C

The following is a list of valid ~zlues that can be written out to port 71h for controlling the heater.
OOh Turn heater off.
Olh Turn heater on.

-13403~0 , I
Vacu~ Controll~:
Port: 74h In~ector Val~es 75h 5 inch Ballast Level - The vacuum level $n the ballast tanks is read through port 75h. The level of the ballast ~s ma~ntained by reading the vacuum level, and then bleeding the tank up or down to adjust it.
The bleed valves are controlled by writing a bit pattern to port 74h. Each b$t position in the byte represents one of the-valves.
The following derivation converts tbe A/D reading of the ballast level to vacuum in psi.
O to 5 Vin ~ O to 15 ~nchec 255 bits - 15 tnchec 1 bit - .1197 ~nches o f mercury ~ADC value ~ 119.7) / 1000 - inches The following is a list of valid values that can be written out to the valve port (74h).
FBh Turn On bleed-up valve.
F7h Turn On 81eed-down valve.
EEh Hi-Vacuum ln~ector.
EDh ~o-Vacuum in~ector.

51 13~039~

E~ Vol~age Controller:
Port: 70h High Voltage Control 77h High Voltage Current The high voltage power supply uses a O to 10 V input to produce a O to 30,000 V output. The DAC v~luc for the output voltage is written to port 70h. The current across the capillary is read at port 77h.
The following i8 derives the equation to convert a voltage setpoint into an DAC value:
O to 10 Vin - O to 30,000 VOut 1 Vin ~ 3000 Vout 255 bits ~ 10 Vin 1 bit ~ 0.03921S6 Vin 1 Vin_ ~ 0.0392156 Vin_ 3000 Vout ' X Vout X Vout ~ 117.65 Vout 117.65 V~ut per 1 bit setpoint / 117.65 VOut - DAC value NOrE: setpoint i5 in increments of 1000 V
~ his derivation converts the high voltage ADC reading to a value in Volts.
O to 4.95 Vin ' O to 0.33 mA
255 bits ~ 4.95 Vin 1 bit ~ 0.0194117 Vin ~ he next derivation converts the current ADC re~ng to a current in yA.

E~ pol~r~ty RelAY
Port: 74h value of :Oh Engaged relay (on) gives a minus charg- to the receive buffer side of the cathode. Disengaged relay (off) gives a plus charge to the receive buffer s~de of the cathode.

A

~ 52 1 1340390 Ç~ ~ent RelAy Port: 74h value of 80h Th~s is the port used to turn on the Integrator.

H. ~P~ter ~lao RelaY:
Port: 74h value of 40h ~ his port is used to let heat out of the box if it goes lS-C
over the desired temperature.

VTTT . State Notes 1~ Tnterlocks: -2. ~nual~ze TemDerature:
3. Vacuu~ Wash:
4. ~-Alize R~-ffP-:
5. Mark~r ~n~ect General Operat~on:
C. Sample ~n~ect General Operation:
7. sntect Vacuum:
8. Tn~ect Voltaqe:
USQ ~/Mod (nl,n2,n3 - ~,n4) -~ nl~n2/n3 Round up or down using remainder : setpt -> Code ~ <setpt - code~ ) Now ro~n~e~ d/a setpiont code is on top of stack :S fetc~
setpt -~ code dup code! port p! ; Send it to D/A
A/D ~alue (00-FF) ~ 13 ~constant) ~ ~o~e decimal one place to.
left and get current reading ln micro amps. ~ 0-330 ~A
t Zero ~vent ~.v~ess~n~:
10. ~nteqrator:
11. Start Auto~atic ODerat~on:
12. ~n~ Auto~at~c ODer~t~on:
13. Start~nd Manual Operat~on:
A

54 13~o3 14. ~otate Vials:
Enter: Value o~ ~ial desired a. At down yes/nQ
~ Q 1. A~rm error exit ye~ b. Get value of ~ial pos$tion c. ~urn on Motor d. Read rough LED's wait for match ye~/nQ
o 1. loop here till match a. get t~e ~ save first entry b. see if time > than 2 ~ec. ye~/nQ
nQ loop ~ look for ~atch yes Alar~ error exit yes e. Read Exact LED wait for ~atch yes/DQ
no 1. loop here till match a. get time ~ save first entry b. see if time > than 1/8 sec. yes/no no loop ~ look for match yes Alarm error ex~t ye~ f. Turn Motor off g. Clear stack of ti~e data h. Continue next ~equence Exit: At vial location desired Error Exit:
a. buzzer on b. display error ~essage t See display routine c. go to Idle mode t See Abort Seqpence 15. n~wn V~ls:

16 . ~D V~ als 17. ISR:

18. Ronnd Rob~n Osera~on:
a. Software ~nterloc~ Mirror Check ~ Future when can read ports) b. Voltage A/D
c. Vacuum A/D
d. Te~perature A/D
e. Absorbance A/D
f. Status display update g. Chem$stry h. Run Setup i. Edit j. Print k. RS-232

Claims (3)

1. A method of performing capillary electrophoresis comprising:
introducing an electrophoretic medium into a capillary;
introducing sample into the capillary;
applying an electric field to the capillary to cause electrophoresis;
adjusting the temperature in the capillary to vary the pH to cause differential separations in the sample during electroyhoresis;
detecting the sample introduced into the capillary.

2. The method of Claim 1 wherein the step of adjusting comprises running the electrophoresis for a first period of time at a first temperature and then running the electrophoresis for a second period of time at a second temperature.

3. The method of Claim 1 wherein the step of adjusting comprises changing the temperature in the capillary during electrophoresis according to a preselected temperature profile.