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so as to alter its direction, as shown in the diagram, and as will be after-
wards referred to. Placed anteriorly to the lower half of Albrecht's body
is the small Xichol's prism d. Corresponding to the upper part of the glass
body is a composite glass plate e, with perfectly parallel sides. This plate is
formed by cementing together two glass wedges, of Avhich one is of clear glass
and the other of smoke-tinted glass, and can be moved from side to side by
means of a special arrangement. According to the position of this plate it
will absorb more or less light. The purposes of these various parts are
sufficiently obvious froin the diagram ; aa represents the absorption-trough for
containing the coloured liquid to be spectrophotometrically investigated. In
the lower half of the trough is seen the Schulz's cube (5) ; r and ?•' represent
two parallel beams of light falling on the anterior surface of the trough. The
lower beam (?■) traverses in its path the ISTichol prism (i/), and is polarised ;
falling then on the adjacent surface of the parallelopiped, it is deviated so
as to fall upon the upper half of the slit. The upper beam may or may not
meet in its path the composite plate c previously referred to, and to which
reference will again be made. This beam is so deviated as to fall upon the
lower half of the slit. After traversing the structures just described, two
beams of light fall upon the slit — a polarised beam on the upper half and a
non-polarised beam on the lower.

2. Tlie telescope. — A very ingenious arrangement, which is indicated by
a separate drawing in the centre of Fig. 31, permits of the precise
position of the telescope in reference to the prism being determined, and
consequently of the most accurate determination of the position of any line
in the spectrum. The reader is referred for details to Professor Hiifner's
original description. At the distal end of the telescope is the object glass,
next to it is a Xichol's prism, the rotation of which is measured on a graduated
circle by the help of a vernier. In the focal plane of the eyepiece is a
modification of Vierordt's eyepiece slit, permitting of any determined spectral
region being exactly isolated. For further details as to the construction and
adjustment of the spectrophotometer, the reader is referred to the original



SPECTROFHOTOMETRIC CONSTANTS. 223

sources of information. It is absolutely essential to work with Hiifner's
spectrophotometer in a perfectly darkened room.

Before commencing photometric measurements, the observer will ascertain
whether the analysing Nichol is in the position in which it allows the polarised
beam to pass unabsorbed. He will then fill the absorption-trough, and isolate
and measure the spectral region for which the extinction-coefficient is to
be determined.

On now looking through the eyepiece two spectral strips Avill be seen,
separated by a sharp horizontal line ; these spectral strips will be of unequal
brightness ; the upper, being a portion of the spectrum of the polarised
beam, will be much less luminous than the lower. The composite glass
plate in front of the slit of the collimator is now moved inwards in the
direction of the beam of the unpolarised light, so as to diminish its intensity,
until the upper and the lower spectral strips appear of precisely the same
brightness.

The trough containing the coloured liquid under investigation is now
brought into position, the upper surface of the glass cube in the trough being
placed about 1 mm. below the plane passing through two horizontal angles of
Albrecht's glass body. On now examining the spectra, it is at once seen that
the lower of the two is darker than the upper. The analysing Nichol is then
carefully rotated until equality in the intensity of the two spectral strips is
attained ; the angle through which the prism has been moved is then deter-
mined ; several, say five, sets of readings being made in two opposite quadrants
of the large divided chxle. The mean of these readings gives the value

of (/).!

The spectrophotonietric constants of oxyhsemoglobin. — It was
previously stated that it is usual to determine the photometric constants
of colouring matters in two spectral regions, tliom regions leing chosen
in lohich the variations in the absorption of light are most rapidly affected
hy variations in the concentration of the colouring matter.

The reasons for determining in the first instance at least two values
for A (which we shall distinguish as A and A!), and subsequently, each
time that a determination is made, ascertaining the value of e in the
same two regions (the two extinction-coefficients being distinguished
as e and e', or in the case of oxyhcemoglobin as c^, and e,,') are the
following: — (1) If we know the value of A and A' for any body, we are
able to make two independent estimations when determining the
concentration of a solution of tlie same body of unknown strength, the
one estimate acting as a check on the other. (2) The knowledge of the
value of A and A', for each of two colouring matters co -existing in
solution, is a necessary condition to being able to determine spectro-
photometrically the amount of each constituent when occurring together.
(3) In the case of oxyh?emoglobin, haemoglobin, and CO-hcemoglobin, the

quotient - is absolutely characteristic of each substance, and affords a

valuable check on the purity of the colouring matter in solution and on
the accuracy of the analysis.

Hiifner's most recent determinations ^ of the spectrophotonietric
constants of oxyhaemoglobin, made with his perfected spectrophotometer,
have led to the results shown below. The two values of A are, as has

^ Hiifner's spectrophotometer is constructed by, and can be obtained from, the original
maker, Herr Eugen Albrecht, Universitiits-Mechaniker in Tiibingen.

^Hiifner, "Photometrische Constanten des Oxyhamoglobins, "^rcA./. Physiol., Leipzig,
1894, Physioh Abth., S. 134 et seq.



224



HEMOGLOBIN.



been previously stated, in the case of oxyhcemoglobin, distinguished by
the symbols A^ and A^^ ; the first has been determined for the spectral
region which lies between the two bands a and ^ of oxyhaemoglobin,
and is limited by >. 554 and a 565 ; the second has been determined for
the spectral region which corresponds to the darkest part of the second
(/3) band of oxyhsemoglobin, and extends from >. 531-5-X 542-5.



Spectrophotometric Constants of OxyluBmogloljin ^ (Hufiier).



Limits of Spectral Region iu which
£„ and £'„ were determined.



Absorption relation A^ and A' ^ corre-
sponding to the two regions.



(iZofe^) . . X554-\565
(iJofe'o) . X 531 -o-X 542-0



^0



0-002070
0-001.312



From the above constants we are able, as has been shown (see p. 215),
to determine the percentage of haemoglobin in the blood with surprising
accuracy. The further use of these constants will be referred to in
explaining the mode of determining the relative amounts of hcemoglobin
and oxyhcemoglobin coexisting in any sample of blood.

We have now to consider in some detail the light which spectro-
photometry has shed on certain questions which possess great interest
to the physiologist, and which have up to a certain point been already
discussed in this article.

Hlifner and his pupil v. Noorden long ago noticed that the quotient

" which is the same as — , was remarkably constant, not only in

the blood of animals of the same species, but in all, however widely
separated in the animal scale.^ Subsequent researches by Hlifner and
his pupils, carried out with a much more perfect spectrophotometer
than the one employed by v. Noorden and himself, more than confirm
the earlier results in so far as the constancy of the quotient is
concerned.

If the defibrinated blood of any animal, diluted with 150-160 parts
of 01 solution of NaOH, or a solution in the same dilute NaOH of
crystals of oxyhcemoglobin of approximately equivalent concentration,
be thoroughly oxygenated by shaking with air and the values of Sq and

g'o be determined, it will be found that the quotient — will vary very

So

sUfhtly from 1-580. In very few determinations, out of a large number,
was it as low as 1-578. vSo soon, however, as the blood commences to
undergo any change, as, e.g., a partial conversion into methsemoglobin,
the coefficient is lowered.



^The values of A^ and A\ given above differ materially from those which had been
assigned to tliem previously by Hlifner and his pupil v. Noorden as a result of researches
carried out with Hiifner's earlier and much less perfect spectrophotometer, and employing
hfemoglobin whicli had been frequently rera-ystallised.

2 V. Noorden's observations included the blood of man, the dog, the cat, the rat, the
guinea-pig, and the owl. " Beitrage zur quantitativen Spektralanalyse, in bcsondere zu
derjenigen des Blutes " (aus d. Lab. d. Prof. Hlifner in Tiibiugeu), Ztschr.f.2?hysiol. Chcm.,
Str'assburg, 1880, Bd. iv. S. 9-35.



THE PHO TO GRAPHIC SPE CTR UM. 225

From the extraordinary constancy of this quotient some interesting
conchisions may be legitimately drawn. (1) The constancy of the
quotient in all animals affords presumptive evidence, amounting to
absolute proof, that the iron-containing molecular group existing in
hcemoglobin, upon which its colour, its light-absorbing power, and its
capacity to combine with 0, GO, and NO depend, is identical in
all animals. The truth of this hypothesis is borne out by many
weighty facts, e.g. the identity in chemical composition (as revealed
by analysis) of the iron-containing products of the decomposition
of haemoglobin, whatever its source ; the constancy in the propor-
tion of and CO which can combine with 1 grm. of hemoglobin
of different animals. (2) The constancy of the quotient (whether
solution of crystalhsed hsemoglobin, or an alkaline solution made by
diluting defibrinated blood with O'l per cent. vol. of Na(OH), or a
liquid holding intact blood corpuscles in suspension, be investigated),
shuts out the possibility of more than one colouring matter existing
in the blood. It renders absolutely untenable the views of Bohr,
who has assumed the existence of several haemoglobins, possessed
of different powers of combining with oxygen ; and utterly disproves
Hoppe-Seyler's hypothesis that the colouring matter of the corpuscles
is distinct from heemoglobin so as to deserve a special designation of
arterin or phlebin, as the case may be.

(&) The photographic spectrum. — In the year 1878 the late
Professor J. L. Soret, of Geneva, in his first memoir on the absorption
of the ultra-violet rays of the spectrum by diverse organic substances,^
announced the fact that diluted blood, when examined with the aid
of a spectroscope provided with a fluorescent eyepiece, presented in
the extreme violet, between Frauenhofer's lines G and H, an absorp-
tion-band which appeared to him to be slightly shifted towards the
less refrangible end of the spectrum, when the blood solution was
saturated with carbonic oxide. Soret subsequently^ confirmed the
accuracy of the above facts, employing the photographic method in
his experiments, though he published none of his photographs. Since
the date of the publication of Soret's short notes on this subject,
d'Arsonval ^ has independently, and without referring to Soret's observa-
tions, described anew the extreme violet absorption-band of the blood-
colouring matter, but without adding to the facts discovered by the
Swiss observer.

The complete absence of all reference to Soret's scanty but
interesting and suggestive observations, in text-books and treatises
on physiology and physiological chemistry ; and the fact, which my
own observations soon elicited, that the absorption-band of Soret is
even more distinctive of the blood-colouring matter than the absorp-
tion-bands in the visible spectrum which have hitherto engrossed the
attention of observers, led me to study this absorption-band in more
detail in haemoglobin, its compounds and principal derivatives.* I

^ J. L. Soret, " Recherches siir I'absorption des rayons iiltra-violets par diverses
substances," Arcli. d. sc. j)Tiys. et nat., Geneve, 1878, pp. 322, 359.

2 Soret, ibid., 1883, pp. 194, 195, 204.

^ A. d'Arsonval, Arch. depJiysiol. norm, etpath., Paris, 1890, Ser. 5, tome ii. pp. 340-346.

* A. Gamgee, "On the Absorption of the Extreme Violet and Ultra- Violet Ra3's of the
Solar Spectrum by Hsemoglobin, its Compounds, and certain of its Derivatives,"
Proc. Roy. Soc. London, 1896, vol. lix. p. 276.
VOL. I. — 15



226 HAEMOGLOBIN

propose that the band in the extreme violet should henceforward be
distinguished as the band y, or the band of Soret, in the spectrum of
oxyhtemoglobin.

Methods of demonstrating the land of Soret. — The hmits of visibility
of the solar spectrum correspond, as usually stated, with the H group of
Knes ; here lies the arbitrary boundary which separates the extreme
violet from the ultra-violet properly so called — that region which
we can only see by interposing fluorescent media in the path of the
rays {e.g. a fluorescent eyepiece), or by allowmg the spectrum to fall
on a fluorescent surface — the region which is best studied by the aid of
photography.

Although Soret's band lies at the limit, but yet within the boundaries,
of the visible spectrum, it is impossible to see it with the ordinary
spectroscope, i.e., unless this be provided with special devices. It has
already been stated that it can be seen with any spectroscope, if we
substitute a fluorescent for the ordinary • eyepiece ; a cell containing
a dilute solution of sescuhn must, however, be substituted for and
placed in the position of the uranium glass plate of the eyepiece,
uranium glass fluorescing most feebly in the light of the spectral region
where the absorption-band under discussion is situated. It was, indeed,
with the aid of his fluorescent eyepiece that Soret first discovered this
band, though d'Arsonval asserts that it is impossible to see it in this
way. Observations with the fluorescent eyepiece are, however, difficult
and require experience. Still more difficult and unsatisfactory is the
method, also suggested by Soret, and lately published as an original
suggestion by d'Arsonval, of rendering this band visible by interposing a
blue glass between the eye and the spectroscope. If the light be very
intense the band is just perceptible to a person who is already
acquainted with its position and characters through other methods of
observation.

In order to demonstrate Soret's band and the absorption-bands in
the ultra-violet of derivatives of the blood-colouring matter, I projected
the spectrum of sunhght or of the positive pole of the electric arc on to
a fluorescent screen, similar to those which have since come into
common use in observations made with the X or Eontgen rays,
i.e. a screen made by coating a white surface, such as cardboard, with
barium platinocyanide.

In order to render absorption - bands of coloured hquids in the
extreme violet and ultra-violet beautifully visible by this method, it is
essential, however, to open the slit which intervenes between the
source of hght and the collimating lens very widely. In the highly
luminous spectra thus obtained, though none of the spectral lines
are visible, except perhaps H and K appearing blurred and indistinct,
absorption-bands appear with remarkable distinctness and sharpness.
The method is valuable, not only for purposes of demonstration, but
for making preliminary observations prior to having recourse to
photography. By its help I ascertained with correctness the position
and characters of the extreme violet and ultra-violet absorption-bands
of the acid compounds of haematin, of methtemoglobin, of hremato-
porphyrin, and of tm'acin. In no case where this method yielded
negative results, was the presence of a band afterwards demonstrated
by photography.

As few physiological laboratories possess a perfectly darkened optical



THE PHO TO GRAPHIC SPE CTR UM. 227

room provided with a heliostat for projecting a beam of sunlight into it,
the following simple arrangement/ which requires merely an electric
arc lamp and an ordinary laboratory spectroscope of the Bunsen type,
may be adopted.^ The telescope of the spectroscope is removed, and
a beam from the + 'pole of the arc is allowed to fall on the slit of the
collimator. The spectrum is focussed on a fluorescent screen, then the
slit is opened very widely. If the spectrum be a continuous one
(which is the case if it be that of the positive pole of the electric
arc), the coloured solution is then interposed in the path of the beam
falling on the slit.

The 'position cmcl limits of Soret's hand. — Defibrinated arterialised
blood, diluted with from 400 to 600 volumes of distilled water, or still
better with a similar amount of 0"1 per cent, solution of sodium hydrate,

G ^ HK L M N




Fig. 33. — The photographic spectrum of haemoglobin and oxyhsemoglobin.

furnishes solutions (containing about 1 part of oxyhaemoglobin in 3000
and 1 part in 5000 respectively) of a concentration suited for
photographic investigations of the spectrum. With solutions of this
strength (a stratum 1 cm. thick being placed in the path of the beam
falling upon the slit of the collimator) Soret's band can be studied
to perfection, though it can be well seen with solutions much more
concentrated and much more dilute. The appearance and position of
Soret's band in the spectrum of oxyhaemoglobin are shown in Fig. 33
along with that of reduced haemoglobin.

Within fairly wide limits of concentration (the stratum examined
being invariably 1 cm. wide), the limits and characters of Soret's band

1 I employed this simple arrangement in demonstrating these bands in the violet and
ultra-violet to members of the Internat. Physiological Congress, Bern Meeting, September
1895.

" Direct vision spectroscopes cannot be used, the absorption of the ultra-violet rays being
very great in these instruments.



2 28 HAEMOGLOBIN.

remain very constant, the increase in the amount of oxyhtemoglobin
influencing more the intensity of the band than its width. In the case
of solutions containing approximately the proportion of oxyhpemoglobin
above mentioned {i.e. from 0-20 to 0-33 parts in 1000), the spectral
region between F and G- is absolutely unshaded. Soret's l3and is then
seen, extending from X 404— >. 434 ; i.e. it occupies the greater part of the
spectral region intervening between G- and H ; the edges, however,
uniformly shade away as far as these lines.

By examining a series of photographs of spectra obtained by inter-
posing solutions of oxyheemoglobin of very different concentrations, I
have determined that the mean ray absorbed does not, as Soret thought,
coincide with h (a 410-1), but is decidedly on the red side of that line,
corresponding to X 414.

When the concentration of the solution of oxyhemoglobin increases,
the width of the band very slowly increases. Its less refrangible border
never passes beyond G ; as the solution becomes highly concentrated,
the band widens perceptibly, and it does so in the direction of the ultra-
violet. With a solution made by diluting 1 volume of blood to the volume
of 250 (water or 04 per cent, solution of Na(OH) being employed as the
diluent), the absorption-band, though much more intense than with the
more dilute solutions, retains almost the same boundaries, its shadowy
borders approaching, but not passing beyond, G and H. AVith a
solution containing 1 part of blood in 100, the appearances differ
remarkably from those previously referred to. The solution is
transparent for light from F to nearly G ; it transmits light with
difficulty from L to N (a 381-9-X 350-01) ; the remainder of the ultra-
violet is completely absorbed. A solution containing 5 per cent, of
defibrinated blood (or about 6-5 parts of oxyhemoglobin in 1000 parts)
absorbs the whole of the violet and ultra-violet regions of the spectrum,
with the exception of a region between F and G, but nearer the former
(X 460-X 490).

It remains to be considered with how dilute a solution of oxy-
hsemoglobin a photographic record of Soret's band can be obtained.
Examining a stratum 10 mm. broad I have obtained definite results,
when the solution contained somewhat, but not much, less than 1 part
of oxyheemoglobin in 10,000.

No colouring matter yet investigated exhibits the intense absorption-
band between G and H which is characteristic of hemoglobin and its
compounds. Several substances (carmine, picro-carmine, and the colouring
matter of alkanet root) exhibit absorption-bands in the visible part of
the spectrum which bear a superficial resemblance to those of oxy-
hemoglobin. The spectrum of none of these colouring matters exhibits,
however, any absorption in the extreme \dolet or the adjacent ultra-
violet.

The researches which I have conducted have shown that the band of
Soret depends on the iron-containing group existing in the hemoglobin
molecule, yet not upon its iron. The variations in character and position
which this band exhiliits in the various compounds and derivatives of
hemoglobin will be referred to under eacli.



H^MO GL OB IN {RED UCED H^MO GL OB IN). 2 2 9

HAEMOGLOBIN (KeDUCED HEMOGLOBIN).

Synonym, "Pukple Ckuokin."

Historical note. —Fully two years had passed since the date of Hoppe's
publication (1862) of his observations on the spectrum of blood, before it was
shown that the oxygen v/hich enters into combination with hfemoglobin has
a fundamental influence on its spectrum. It was on the 16th of June 1864
that Professor Stokes ^ communicated to the Royal Society the interesting
observation that when diluted blood is treated with certain reducing agents,
the colour of the liquid and its spectrum undergo remarkable changes ; the
former loses its bright red appearance, becoming darker in tint, whilst the
absorption-bands a and /3 are replaced by a single band which we may
designate the band y, which appears less deeply shaded and with less defined
edges, and which extends from D nearly to E. If, now, the solution which
exhibits this spectrum be shaken with air or oxygen, the single band at
once gives place to the two original bands, whilst the liquid reacquires
more or less of its primitive florid-red colour. The process of reduction
and oxidation may be repeated many times in succession.

From his experiments, Stokes concluded that "■the colouring matter ofUood,
like indigo, is capable of existing in two states of oxidation, distinguisliahle by a
difference of colour and a fundamental difference in the action on the spectrum.
It may he made to pass from the more to the less oxidised state by the action of
suitable reducing agents, and recovers its oxygen by absorption from the air." ^

The researches of Magnus, Lothar Meyer, and Claude Bernard had shown
that the blood holds in solution an amount of oxygen greatly in excess of that
which could exist in a state of simple solution, but that this oxygen exists m a
condition which permits of its being extracted from the blood by boiling in a
Toricellian vacuum, as well as by the action of carbonic oxide. Hoppe-Seyler,
having succeeded in crystallising oxyhsemoglobin, and, by means of its optical
properties, having identified it with the colouring matter as it exists in the living
blood, was able to show that a solution of crystallised oxyhsemoglobin behaves
towards reducing solutions in the same manner as diluted blood; that, like blood,
it yields oxygen when boiled in vacuo, and that the blood-colouring matter thus
deprived in vacuo of its loosely combined or respiratory oxygen manifests the
absorption-band which had been described by Stokes as the result of reduction.

The further steps in the growth of our knowledge of reduced haimoglobin
will be more conveniently referred to in discussing the chief facts with which
we are acquainted relative to this remarkable body.

Methods of effecting the reduction of oxyhsemoglobin to reduced
heemoglobin. — In nearly all experiments on the reduction of oxyhsemo-
globin, diluted blood may be substituted for a solution of the pure blood-
colouring matter, it having been shown by the spectrophotometric and
chemical researches of Hllfner that, both in respect of their power of
absorbing light and of the influence of reducing agencies upon them, the
two solutions possess identical properties. Instead, however, of employ-
ing pure distilled water as a diluent, it is advisable to use, according to
Hiifner's plan, a 01 per cent, solution of sodium hydrate. A diluted



Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 32 of 147)