TIBS - October I979

Build your own Amino Aiiid Analyzer

* Rapid 42 Minute Analyses

* Latest Microbore Single Column Method

* Ninbydrin or Fluorescent Detection

* Save 60% or more

* 100 picomoles full-scale sensitivity

* Analyze Peptides, Polyamines

You’ve heard it can be done. . . and it’s true! With the parts and instructions we provide, you can easily assemble this sensitive and rapid analyzer right in your own laboratory! The result is a state-of-the-art instrument that will satisfy every requirement-at a most reasonable price. Fully automatic, easy to operate and reliable, this modern instrument is suitable for peptide finger- print studies and polyamine analyses. as well as for routine assay of amino acids in peptide hydrolyzates and biological fluids.

Microbore single-column technology is combined with either ninhydrin or Fluoropa (o-phthalaldehyde) detection of effluent solutes to provide greater sensitivity and more economi- cal operation than can be obtained with an! other amino acid analyzer on the market. Peptide hydrolyzate analysis time is 42 minutes with a 5 minute resin bed regeneration step between samples; typical eluent pump pressure is 44 atrn (650 lb inV2).

A preassembled, solid-state digital program- mer controls all machine functions including regeneration. Automatic sample injection, avail- able as an option, permits as many as fifteen peptide hydrolyzate samples to be analyzed with the instrument unattended. An integrator can easily be added at any time; because of the instrument’s inherent baseline stability, a relaw tively simple integrator works well.

Complete instructions are provided. Nt) special electronic or mechanical skills are re- quired for assembly: 1 or 2 days are required. The intimate knowledge of the analyzer’s operation gained during assembly makes owner mainte- nance simple. rapid and inexpensive. Should con- sultation be needed at any time, it is as close a$ your telephone: our Technical Service Depart- ment will help you assemble and maintain the instrument or make modifications for special applications. For this service. kit owners can call us collect in the USA and Canada.

Whether you have already decided to purchase an analyzer-or until now didn’t think you could afford one-now is the time to reassess your research plans to include the Dionex Amino Acid Analyzer Kit. Call or write today for complete information.

Assembkd Analyzer. Open framework design greatly facilitates access to components and allows flexibility of format for specialized applications. Preassembled solid-state programmer is on top shelf above level of eluent reservoirs. Chromaropraph! column IS made of stainless steel and is electrically heated.

100 PICOMOLES FULL SCALE Analyzer: Dionex Amino Acid Analyzer Kit

R&in: Dlonex DC-SA Catlon Exchanger

Resin Bead Dlameter: 6.0 r 0.5 /On

Resin Bed Dimenslonr: 0.4 cm jl x 13 cm long

Resin Bed Temperature: 45C+65C at 12 mmutes

Eluents: Dlonex Femto-Buffer System 1

Eluent Flow Rate: 16mL h ’

Eluent Pump Pressure: 44 atm (650 lb in-*)

Callbratlon Sample: 100 picomoles each residue

Effluent Solute Detectlon: Fluoropa (o-phthalaldehyde.

Dionexi

MINUTES 0 10 20 30 40

42 Minute Rptide Hydrolymte Analysis, heretofore possible only on the most expensive analyz- ers, is easily obtained with Dionex Kits. Kits are available with conventional ninhydrin or new fluorescent detection method. Note excellent resolution and symmetrical peak shapes charac- teristic of optimal performance liquid chromatographic systems.

Dionex Cowration

DtONEX 1228 Titan iay, Sunnyvale, CA 94086 Telephone (408) 737-0700 Toll-free ,n Continental USA (except Cahfoma) (8Cil) 538-1882

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TIBS - October 1979 N 242

values of /3 are needed, mere correction for lamp intensity and background variation, which can be achieved in a double beam instrument, will not suffice.

PA calorimetry Besides its use as an analytical tool, two

other applications of PA techniques are emerging - study of thermal properties of solids and liquids, and time-resolved calorimetry of photoinduced processes (the latter is discussed below). Calorimetry is used quite commonly in the study of biopolymers and liquid membranes. Lately, photocalorimetry and photother- ma1 spectroscopy on chemical systems have been described, and a.c. calorimetry (use of periodic heat pulses) has been applied to biological systems. PA calorimetry is essentially modulation photocalorimetry, and as such it resembles photo- and a.c. calorimetry, but uses a much simpler set-up and is capable of faster detection. It com- bines the possibilities of modulation excita- tion spectroscopy (study of kinetics of light-induced processes) and those of photocalorimetry. Looked at in this way it is just the frequency domain equivalent of flash calorimetry [3]. Interestingly, the restrictions on a.c. calorimetry are similar to those dictated by PA saturation, though it should be noted that PA calorimetry on samples that are saturated is quite possible. The advantage of indirect acoustic over direct thermal detection arises mainly from the slow response of thermistors.

A simple chemical example Because the PA signal depends on the

formation as well as on the decay of excited states, and results from thermal decay only, the occurrence of any other decay process (luminescence, photochemistry) will de- crease the PA signal. If such other proces- ses have decay rates matching the chopping frequency of the instrument, we can ‘catch’ their intermediates or products. In this way

’ @@@I in Hex& '

/' .'

A'

\‘\

/ /'

/' ._--_-'

Frequency (Hz)

Fig. 2. Chopping frequency dependence of PA signal (at 340 run) of 1 mM solution of anthracene in hexane, and l-PA signal (=photochemical loss), which is that part of the absorbed energy, not converted into heat. Photochemical loss (left-hand scale, dashed line) PA signal (right-hand scale, solid line). ‘I 0’ on the lefi- hand scale corresponds to -25% loss.

quantum yields, average lifetimes and Z ---! 7”‘711---?~ - -- enthalpy changes in luminescent and ‘5 i t-i. halobium; intact cells

photochemical systems can be determined [4]. Fig. 2 illustrates this in the form of a

b 6kc--‘,k 2 Y-*. b.

frequency spectrum which, at high fre- .p $ 5k ‘t quencies, reflects the energy stored in, and a N-X quantum yield of formation of the triplet 3 4c -9

H state of anthracene. The increase in PA $ \ i cq signal (decrease in loss of signal) with $ 3

,’ ‘._E+ -‘

decreasing frequency is caused by the g thermal decay of this triplet state. At low 2 10 +

r W---“--’ 500

frequencies only fluorescence contributes Frequency (Hz) to a loss in the signal (no loss = total ther- Fig. 3. Chopping frequency spectrum of cells contain-

ma1 decay a optical absorption signal). ing purple membrane at 565 nm, of -1 mm thick pellet

To obtain absolute values it is necessary atpH=7 (-0.2 O.D.); ‘3’arbitrary to&correspond to

to calibrate the PA signal in energy units -50% heat dissipation. The step at 200 Hz (-1200 radlsec) corresponds probably to the decay of the

(see below). If irreversible photoprocesses M 412 photointermediate. A photochemically inactive take place, changes in p need to be taken sample gives a smooth, nearly straight line. (H. Garty et

into account. In such a case it is possible to “.,unpuhlished results.’ get an action spectrum for the photo- dependence of heat dissipation was process. The ideal situation for calibration obtained from a comparison of the PA and is obtained when conditions can be defined optical absorption signal, yielding values under which all the excitation energy goes for the energy which is effectively stored at to heat (PA signal cc input energy). various chopping frequencies [8].

Photobiological uses Fig. 3 illustrates the frequency depen-

dence of the PA signal from cells contain- In photosynthetic systems, applying an ing purple membranes. The steps in this

intense, continuous light beam will saturate spectrum indicate intermediates storing the photoreactions so that all additional different amounts of energy with various excitation energy will be converted into efficiencies and several of them can be heat; this method of calibration has associated with known photointermedi- been used in a study of Chromatium ates. chromatophores by flash calorimetry [3]. Calibration of the signal is a major prob- PA studies on lettuce chloroplasts showed, lem of quantitative PA calorimetry, but as expected, that the largest fraction of several approaches exist, besides those input energy is stored when -680 nm light mentioned above for PA signals in general. and high modulation frequencies are used. Data obtained at very low frequencies can In experiments with DCMU-poisoned sometimes be taken as approximate chloroplasts evidence was obtained for absorption data (= total heat dissipation) incomplete blocking indicated by an for rapid cyclic processes, and then provide energy leak at high frequencies [5]. an ideal method for calibrating heat dissi-.

t The theory of the dependence of the PA pation spectra, as they are obtained under signal on the modulation frequency has the same conditions as are the high fre- been worked out for some simple chemical quency data. Alternatively, the PA and systems as well as for the more complicated optical absorption signals of a reference photosynthetic one [6]. with known optical properties, and thermal

A simpler subject for study is the photo- characteristics close to those of the sample, cycle of bacteriorhodopsin-containing car1 be compared with those of the sample purple membranes of Halobacterium to yield the absolute fraction of absorbed halobium and its associated proton pump. light going to heat. A preferred way is to Flash calorimetry has been used to detect incorporate the reference material into the volume changes (correlated with proton sample or, better still, to use a signal from movements) as well as enthalpy changes the sample that is not connected with its during the cycle 171. Because a cell corn- photoactivity (this is the method used for pletely filled with liquid was used, the Fig. 3). From the theory of photoacoustic effects of volume and enthalpy changes had calorimetry [6] several graphical extrapola- to be separated. Several purple membrane tion methods for absolute energy calibra- preparations have been studied by PA tion can be derived. The use of an electrical calorimetry. both as a function of heating element in the cell for the calibra- wavelength and of chopping frequency. tion of the PA signal in watts will generally The use of an additional strong continuous be unreliable for biological samples. light beam revealed the light-induced because of the different thermal para- absorption changes. The wavelength- meters involved.

TIBS - October 1979

H+ H+ H’ Ii+ H+ H+

l-j+ H+ Iir

Other uses of PA in photobiology and future trends

The analytical applications of PA will have to compete with fluorescence measurements (for detection), and with increas- ingly sophisticated optical transmission and reflectance techni- ques (for identification).

PA methods can be used to study phase transitions, for which calorimetry is usually employed. If the sample is transparent, PA probes such as black particles can be added.

Investigations of systems in viva show some promise, espe- cially in biomedicine and, possibly, agricultural biochemistry [ 11. PA studies on marine phytoplankton, bacteria, human eye lenses (investigation of cataract), and hydration studies of new- born rat stratum corneum have been reported [2]. The hydra- tion studies may be of particular interest because the depen- dence of the PA signal on thermal parameters is used.

In situ investigations using PA cells that are open on one side may be useful, for example, in studies of UV irradiation of skin for understanding sunburn and photocarcinogenesis. PA tech- niques may also be of value in the study of the mechanism of DNA photodegradation and of the visual process.

Several instrumental developments may extend the applica- tions and improve the sensitivity of PA techniques: use of PA cells with the sample outside the PA chamber but in close con- tact with one of its walls, thus allowing additions to the sample during the experiment; further use of gas-free piezoelectric cells for liquid suspensions; further development of flash tech- niques for high frequency measurements; use of transform and dual wavelength techniques for improved signal to noise, and wider use of the information contained in the phase angle changes.

References Adams, M. J., Beadle, B. C. and Kirkbright, G. F. (1976) TIES I, 278-279 Rosencwaig, A. (1978)Adv. Electron. Electron. Phys. 46,207-311 Callis, J. B. (1976)L Rex Nar. Bur. Stand. Sect. A, 80A, 413-419 Robin, M. B. (1977) in Optoacowtic Spectroscopy and Detection, (Pao, Y. H. ed.), pp. 167-191, Academic Press, New York Cahen, D., Malkin, S. and Lemer, E. I. (1978)FEBSLetr. 91,339-342 Malkin, S. and Cahen, D. (1979) Phorochem. Phorobiol. 29 (4) Ort, D. R. and Parson, W. W. (1979)Biophys. J. 25,341-353 and 353-364 (See also (1978),J. Biol. Chem. 253,6158-6164) Cahen, D., Garty, H. and Caplan, S. R. (1978) FEBS Left. 91, 131-134; Garty, H., Cahen, D. and Caplan, S. R. (1978) in Energetic3 and Structure of Halophilic Microorganisms (Caplan, S. R. and Ginzburg, M.. eds), pp. 253-259, Elsevier/North-Holland, Amsterdam I-

2 243

PHOTOACOUSTICS IS FOR REAL!

This is the EDT Model OAS40O Photoacoustic Spectrometer (or Optoacoustic as it is sometimes called) specially developed for the examination of small solid and liquid samples. A major advantage of the photoacoustic technique is the ability to obtain spectra from different depths and sub-surface layers in a non-homogeneous sample. This is especially relevant in Biology where many samples exhibit layered structures. To this end, the OAS400 is equipped with a wide range of modulation frequencies and features a variahle phase control.

PAS can be used for:

* Structural Investigation * Qualitative Analysis * Thermal Diffusivity Measurements * Film Thickness Measurements * Quantitative Determinations

For more details, prices and demonstrations please call or write.

EDT RESEARCH 14 TRADING ESTATE ROAD LONDON NW1 0 7LU

Tel: 01-961 1477 Telex: 8811955 _ j

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