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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979 Table I. Quantitative Comparison of Sample Preparation Methods Pigment (mmol x 10-5)/in.2a old method new method
pigment
I
1
I
4
8
12
l
l
16 20 Time. rnin
I 2a
32
Figure 2. High-performance liquid chromatograph (HPLC) of Clark y9 soybean chloroplast pigments. p C , , reverse phase. Solvent A = 90% MeOH; soivent B = ethyl acetate. Linear solvent program, 0 % B-50% B (20 min). Hold until elution of carotene. Old method, Figure 2a; new method, Figure 2b. Pigments neoxanthin ( I ) , violaxanthin (2),lutein (3), chlorophyll b (4), chlorophyll a (5),and carotene (6)
disks of leaf with a 27 cork borer. In a typical experiment. 10 disks (one disk from each of 10 different plants) are introduced into a glass tissue grinder and homogenized with 1-2 mL of '707~ methanol. Both the tissue homogenizer and methanol are ice cold to prevent chlorophyll degradation. After addition of 0.08 g celite, the homogenate is drawn into the syringe via the three-way valve, then forced through the Millipore filter into the Sep-Pak cartridge. The homogenizer is washed with an additional 8 mL of 7 0 7 ~ methanol. This wash is also drawn into the syringe and forced through the filter and Sep-Pak. By this time, the flavonoids have been washed from the leaf and through the Sep-Pak; and after heating or hydrolysis may be analyzed by HPLC (3). Carotenoids and chlorophylls are visibly retained on the Sep-Pak. At this point, the carotenoid and chlorophyll pigments may be broadly separated into classes by stepwise elution or completely removed for high-performance liquid chromatography (HPLC) analysis. In the stepwise alternative, elution of Millipore filter and Sep-Pak
neoxanthin violaxanthin lutein chlorophyll b chlorophyll a carotene
1.06 i: 2.70 i 4.00 i 5.75 * 19.45 i 3.82 *
0.04 0.36 0.25 0.50
0.68 0.18
1.00 i 2.14 i 3.84 i 5.79 i 20.49 f 3.74 i
0.098 0.12 0.24 0.31 0.81
0.24
a Mean of three determiiiations of soybean pigments Clark L1, growth chamber grown-3115179. -
with 90% methanol (approximately 5 mL) will remove the oxygenated carotenoids, neoxanthin, violaxanthin, and lutein. A change of eluant to 10070methanol will remove chlorophyll a and chlorophyll b. Finally, carotene may be eluted with acetone. If total recovery of all pigments is desired, one goes directly to acetone solvent. The pigments are eluted into a 5-mL volumetric flask and a sample is injected directly. R E S U L T S A N D DISCUSSION We have examined a number of leaf samples and have compared the results of HPLC analysis for these samples using both our old method and the new procedure of sample preparation. The results were comparable both qualitatively (Figure 2) and quantitatively (Table I). Once the samples are taken up in acetone and stored in the refrigerator, they will keep without decomposition for several weeks. T h e method presented here for sample preparation represents a real savings in time and has fewer manipulations which expose the pigments to light, heat, and oxygen. Although the described method is for chloroplast pigments of higher plants, it should have general application in preparation of biological samples for HPLC analysis. LITERATURE CITED (1) E. H. Davis, "Analysis of Carotenoid Pigments", in "Chemistry and Biochemistry of Plant Pigments", Vol. 2, T. W. Goodwin. Ed.. Academic Press, New York, 1976, pp 38-155. (2) K. Eskins, C. R. Scholfield, and H. J. Dutton, J . Chromatogr., 135, 217 (1977). (3) S. Asen, Hort. Sci., 12, 447-448 (1977).
RECEILZD for review January 18, 1979. Accepted May 3,1979. The mention of firm names or trade products does not imply t h a t they are endorsed or recommended by the C.S. Department of Agriculture over other firms or similar products not mentioned.
Nuclear Magnetic Resonance Spectra Reduction by Electronic Modification of the Recorder on a 100-Megahertz Spectrometer Daniel J. O'Donnell, Stan 0. Sigle, and K. Darrell Berlin" Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74074
Gene
P. Sturrn
Bartlesville Energy Technology Center, Bartlesville, Oklahoma 74003
In the course of several investigations which required extensive NMR analysis, it became apparent that the accumulation of a very large number of standard size (50 X 25 0003-2700/79/0351-1886$01 OO/O
cm) plots of the spectra would present a storage problem. The miniaturization via photoreduction of the full scale spectra to a size more suitable for storage (and for possible repro8 1979
American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
1
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TP2
V ut fo,(
R 2 4 ICK
R 4 6 IOK
PC S o c k e t
-i-
I
n
R23 IK
,
.-.
I
n R 25
I,
I_
R
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I
1
Figure 3. Mounting of the circuit board and switch on the recording console, with switch shown in the “normal” position
:
-
Figure 1. Schematic diagram of the recorder plot reduction modification (bold lines) including a partial diagram of the original circuitry ( 7). Component list: R(a), 10-kR precision Cermet trimmer potentiometer; R(b) and R(c), 5-kR precision Cermet trimmer potentiometer; switch, 3-POT subminiature switch
Flgure 2. Modified y-axis servo motor control board. Note the six wires soldered to the previously unused pin clip connectors in the lower left of the picture; the seventh connection is through existing foil at lower left, from test point 2 to pin 2 at socket
duction for publications or theses) was prohibitively expensive for such a large number of spectra. Photoreduction as a means of miniaturization also suffered from the obvious fact that a full scale spectral printout was still necessary in order to obtain a reduction. Although direct reduction of the spectrum on the recorder could be achieved by setting the amplitude and sweep width accordingly, the method proved time consuming on a large scale. Also, no simple method exists for resetting the upper and lower recorder limits to prevent the pen from overrunning the selected plotting limits. In order to eliminate these restrictions on the direct reduction of the spectra with control circuit of a Varian XL-lOo(l5) spectrometer was made.
This modification provided a simple means to obtain reduced spectral plots of a predetermined size a t a minimum expense of time and cost. The modification alters the gain in the y-axis servo control and changes the upper and lower limits of the y-axis servo control. The new circuits are wired through a %pole, double-throw switch so that the original circuitry can be easily restored for regular plotting. In addition, all of the modifications were designed for quick removal from the original circuit module to simplify repairs and/or replacements.
DESCRIPTION OF CIRCUITRY A N D MOUNTING DETAILS A schematic of the modifications, including a partial schematic of the original circuitry (1 I , is shown in Figure 1, and a picture of the modified circuit board is shclwn in Figure 2. As can be seen from Figure 1,three new circuits (outlined in bold face lines) are formed when the triple-pole, doublethrow (3 PDT) switch is in the “reduce” position. The y-axis gain is altered by eliminating resistors R24 and 1246 from the circuit governing the negative feedback on operational amplifier A2. These two resistors are replaced in the new circuit the recorder, a simple modification of the y-axis servo motor by R(a), a 10-kR precision Cermet trimmer potentiometer. This change lowers the total resistance in this circuit which increases the negative feedback voltage of the amplifier, thus reducing the y-axis gain by an amount dependent upon the resistance value of the potentiometer. The lower and upper electronic stops in the original circuitry are governed by transistors Q8 and Q9 and diodes CR5 and CR6. In the modified circuitry, both Q8 and Q9 are effectively by-passed by resistors R(b) and R(c), two 5-kR precision Cermet trimmer potentiometers, respectively. The level of resistance for these two potentiometers will determine the position of the electronic stops. The three potentiometers were mounted on a small circuit board attached to the main recording console as shown in Figure 3. Also shown in Figure 3 is the position of the subminiature switch. This particular configuration was chosen to prevent the accidental tripping of the switch.
OPERATING CHARACTERISTICS The effective removal of transistors $8 and (29 from the circuits controlling the lower and upper limits might be expected to result in a “softening” of the stops (Le., a high amplitude signal would produce a rapid movement of the pen,
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ANALYTICAL CHEMISTRY, VOL 51, NO 11, SEPTEMBER 1979 ppm
r
180
160
140
I
1
I
BO I
100
120 I
1
60
I -
52 05 00 00
1000 50 0
: Solvent.
PFT-CWL
.
Size. . N A K;
P2/g.
. NA
Gated O f f .
DC.
;
.
DCC13;
35
.
SO.
p/a; SF.
NA;
Offset.
. .
85774 Hz;
PW.
1 0 0 . 1 MHz;
FB.
. 1000 H z ; . 2 Hz;
.
NA H z :
RF.
. NA
W/dB;
T.
.
37 “C;
.
Lock.
NBW.
.
2H ; NA
.
Acq/g.
D5/E.
.
1.0
1000 s
Hz
Figure 4. Reduced size ’H NMR spectrum of o-dichlorobenzene (30%) and acetone (5%) in chloroform-d, plotted on specially designed chart paper. High and low electronic stops have effectively limited the pen movement from overrunning the chart boundaries for the acetone signal (6 2.09). Pen response and recorder sensitivity were high, as evidenced by the detail in the aromatic region and the spinning side bands evident for acetone and TMS
giving it sufficient inertia to carry it past the stop). Although both limits in the reduced mode do show some “softness”, it is not appreciably greater than the “softness” observed in the normal mode with the original circuitry restored. Even though it might be possible to modify the limits and still retain the use of Q8 and Q9, the present modification has the advantage of simplicity, using the fewest number of components to achieve the desired results. In order to obtain a reduced spectrum with plot dimensions of 20 X 10 cm (40% of a normal 50 X 25 cm plot), the sweep width must be changed to a value 5/2 of that normally used. Thus, for a reduction of ‘H NMR spectrum of 1000 Hz, the switch would be set in the “reduce” position, and a plot width of 2500 Hz would be selected either through the recorder console or by computer control if a minicomputer is available for FT mode experiments. T h e dimension of 20 X 10 cm is particularly attractive, since it fits conveniently on an 81/2
X 11 inch sheet of ordinary typing paper. In our laboratory, special recording paper was made a t a cost of $0.03 per sheet (see Figure 4 with ODCB). The upper and lower limits as well as the y-axis gain may be set through the added potentiometers to accommodate any desired sheet size. Finally, the total cost of components was less than $10 by 1979 prices, and the total modification required less than a day to install.
-
LITERATURE CITED (1) Varian Publication no. 87-125-742, “Y-axis Servo Assembly”, Varian Associates, Palo Alto, Calif.
for review April 16, 1979. Accepted May 25, 1979. Work supported by the Department of Energy, Bartlesville Energy Technology Center, Bartlesville, Okla., 74074, under contract number EW-78-A-19-0001. RECEIVED
