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Studies in the preparation of pollen extracts
STTJDIES IN THE PREPARATION
OF POLLEN
EXTRACTS”
H. HAROLD GELFA~TD, M.D.,
GEORGE FI,AM*I, M.D., a. c);. CENTER, AND A. J. HEIFITZ, B.S. NEW YORK, N. Y.
B.S.,
I
N THE extraction of pollens and house dust, Coca1 recommended the use of “alkaline fluid” in preference t,o “buffered saline fluid” hecause extracts prepared of t,he latter were found t,o deteriorate considerably within a few months. When the alkaline fluid was employed, only a relatively slight deterioration of t,he cxcitant occurred. Working with similar preparations, Milford” observed that keeping house dust extract continuously provided with a slight excess of carbon dioxide was an important factor in preserving the specific acl.ivity of the ext,ract, when it was being concentrated by fanning. In allergy laboratories where pollen extracts are prepared with the alkaline extracting fluid as prescribed by Coca, and then filtered through a Seitz or Berkefeld filter by means of suction, one unsatisfactory result occurs. It is the general experience that, although a perfectly clear solution is introduced into the filter, the filtrate frequently returns showing precipitation and containing sediment. IJp to the present time the only way to overcome the use of frothy pollen extract was to allow the preparation to stand until the flakes settled to the bottom, then to decant off t,hc clear solution for use. This is a tedious procedure entailing possible contamination and loss of ingredient, and with it, loss of the specific cxcitant activity of the extracL3 We determined that this precipitation and sedimentation in the solution was caused by the withdrawal of carbon dioxide from the alkaline extracting fluid in the process of the suction filtration. When carbon dioxide was experimentally passed through a frothy solution, it promptly became clear. On the other hand, when carbon dioxide was withdrawn, a Erot,hy cloudy solution again resulted. WC, t,hereforc, recommend filtration by means of carbon dioxide pressure rather than suction, for by the use of this method t.he solutions are always clear. DESCRIPTION OF APPARATUS
A tank of carbon dioxide, to which is equipped with a gauge pressure, is connected by fittings found that in filtration a supply necessary for the production of *From
The
Department
of
Allergy,
which is attached a reducing valve and to control the supply of the gas under to a S&z filt,er under pressure. It was of ten to twenty pounds of pressure is a clear filtrate. By means of multiple Gouverneur 336
Hospital,
New
York,
N.
Y.
GELFAND
ET
AL,. :
PREPARATION
OF
POLLEN
337
EXTRACTS
connections attached to the tank, it is possible to filter into a number of flasks at the same time, according to requirements, but with a corresponding regulated increase in pressure. The rapidity of filtration is directly dependent. upon the pressure released. Since it is evident that the more important factor in the suggested met,hod is the use of posit,ive pressure behind the fluid (in order to prevent the loss of CO,, which occurs when the filtered fluid is subject t,o a negative pressure), our object could have been obtained equally well if pressure was made with air instead of with carbon dioxide. CONCLUSION
The precipitation and sedimentation in pollen and other allergenic extracts which followed vacuum filtration were found to be due to the withdrawal of carbon dioxide during this suction process. Pollen extracts can he rendered permanently clear by filtration through a Seite or Berkefeld filter under pressure of carbon dioxide or air. The use of pressure of this kind eliminates loss of the gas content which occurs during vacuum filtration. Filtration under pressure of carbon dioxide or air is recommended for use to prevent : (1) loss of free and some combined carbon dioxide in the extract; (2) sedimentation and precipitation; and (3) loss of specific excitant content. Further work is being done to determine the amount of specific activity lost in the sediment resulting from vacuum filtration. NOTE : “Buffered Rttline fluid ” IS an extracting The by Coca and commonly used for extraction. posed, are kept in concentrated forms, namely:
50.00 grams 3.63 grams 14.3 1 grams
1. NaCl
KH,PO, Na,HPO, A mixture of distilled lvater. The “alkaline
equal
parts
solution”
2. Carbolic
of thcxse is composrd
two
stock
If
solutions
acid
is diluted
4%
with
4 part,s
0.5 yo 0.275% 0.4 %
the fluid is alkaline to phenolphthalein, carbon dioxide it remains colorless when tested with that indicator. ready for use in tightly stoppered bottles.
is passed through it This solution is then
REFERENCES
1. Coca,
of
of’ :
NaCl NaHCO, Phenol until kept
solution originally recomrnrndetl two solutions of wlrich it is don-
Arthur F., and others: Asthma and Hay Fever in Theory and Practice, Springfield, Illinois, 1931, Chas. C. Thomas. 2. Milford, E. L.: Ibid., p. 101. 3. Gelfand, H. Harold, Flamm, George, and Heifitz, A. .I.: Specific Excitant Activity in the Sediment of Pollen Extracts Obtained by Vacuum Filtration. In preparation.
OF POLLEN
EXTRACTS”
H. HAROLD GELFA~TD, M.D.,
GEORGE FI,AM*I, M.D., a. c);. CENTER, AND A. J. HEIFITZ, B.S. NEW YORK, N. Y.
B.S.,
I
N THE extraction of pollens and house dust, Coca1 recommended the use of “alkaline fluid” in preference t,o “buffered saline fluid” hecause extracts prepared of t,he latter were found t,o deteriorate considerably within a few months. When the alkaline fluid was employed, only a relatively slight deterioration of t,he cxcitant occurred. Working with similar preparations, Milford” observed that keeping house dust extract continuously provided with a slight excess of carbon dioxide was an important factor in preserving the specific acl.ivity of the ext,ract, when it was being concentrated by fanning. In allergy laboratories where pollen extracts are prepared with the alkaline extracting fluid as prescribed by Coca, and then filtered through a Seitz or Berkefeld filter by means of suction, one unsatisfactory result occurs. It is the general experience that, although a perfectly clear solution is introduced into the filter, the filtrate frequently returns showing precipitation and containing sediment. IJp to the present time the only way to overcome the use of frothy pollen extract was to allow the preparation to stand until the flakes settled to the bottom, then to decant off t,hc clear solution for use. This is a tedious procedure entailing possible contamination and loss of ingredient, and with it, loss of the specific cxcitant activity of the extracL3 We determined that this precipitation and sedimentation in the solution was caused by the withdrawal of carbon dioxide from the alkaline extracting fluid in the process of the suction filtration. When carbon dioxide was experimentally passed through a frothy solution, it promptly became clear. On the other hand, when carbon dioxide was withdrawn, a Erot,hy cloudy solution again resulted. WC, t,hereforc, recommend filtration by means of carbon dioxide pressure rather than suction, for by the use of this method t.he solutions are always clear. DESCRIPTION OF APPARATUS
A tank of carbon dioxide, to which is equipped with a gauge pressure, is connected by fittings found that in filtration a supply necessary for the production of *From
The
Department
of
Allergy,
which is attached a reducing valve and to control the supply of the gas under to a S&z filt,er under pressure. It was of ten to twenty pounds of pressure is a clear filtrate. By means of multiple Gouverneur 336
Hospital,
New
York,
N.
Y.
GELFAND
ET
AL,. :
PREPARATION
OF
POLLEN
337
EXTRACTS
connections attached to the tank, it is possible to filter into a number of flasks at the same time, according to requirements, but with a corresponding regulated increase in pressure. The rapidity of filtration is directly dependent. upon the pressure released. Since it is evident that the more important factor in the suggested met,hod is the use of posit,ive pressure behind the fluid (in order to prevent the loss of CO,, which occurs when the filtered fluid is subject t,o a negative pressure), our object could have been obtained equally well if pressure was made with air instead of with carbon dioxide. CONCLUSION
The precipitation and sedimentation in pollen and other allergenic extracts which followed vacuum filtration were found to be due to the withdrawal of carbon dioxide during this suction process. Pollen extracts can he rendered permanently clear by filtration through a Seite or Berkefeld filter under pressure of carbon dioxide or air. The use of pressure of this kind eliminates loss of the gas content which occurs during vacuum filtration. Filtration under pressure of carbon dioxide or air is recommended for use to prevent : (1) loss of free and some combined carbon dioxide in the extract; (2) sedimentation and precipitation; and (3) loss of specific excitant content. Further work is being done to determine the amount of specific activity lost in the sediment resulting from vacuum filtration. NOTE : “Buffered Rttline fluid ” IS an extracting The by Coca and commonly used for extraction. posed, are kept in concentrated forms, namely:
50.00 grams 3.63 grams 14.3 1 grams
1. NaCl
KH,PO, Na,HPO, A mixture of distilled lvater. The “alkaline
equal
parts
solution”
2. Carbolic
of thcxse is composrd
two
stock
If
solutions
acid
is diluted
4%
with
4 part,s
0.5 yo 0.275% 0.4 %
the fluid is alkaline to phenolphthalein, carbon dioxide it remains colorless when tested with that indicator. ready for use in tightly stoppered bottles.
is passed through it This solution is then
REFERENCES
1. Coca,
of
of’ :
NaCl NaHCO, Phenol until kept
solution originally recomrnrndetl two solutions of wlrich it is don-
Arthur F., and others: Asthma and Hay Fever in Theory and Practice, Springfield, Illinois, 1931, Chas. C. Thomas. 2. Milford, E. L.: Ibid., p. 101. 3. Gelfand, H. Harold, Flamm, George, and Heifitz, A. .I.: Specific Excitant Activity in the Sediment of Pollen Extracts Obtained by Vacuum Filtration. In preparation.